Ubiquitin ligase assay

ABSTRACT

The invention relates to assays for measuring ubiquitin ligase activity and for identifying modulators of ubiquitin ligase enzymes.

FIELD OF THE INVENTION

This invention is directed to assays for measuring the activity ofubitquitination enzymes. The invention is also directed to assays foridentifying modulators of ubiquitination.

BACKGROUND OF THE INVENTION

Ubiquitin is a highly conserved 76 amino acid protein expressed in alleukaryotic cells. The levels of many intracellular proteins areregulated by a ubiquitin-dependent proteolytic process. This processinvolves the covalent ligation of ubiquitin to a target protein,resulting in a poly-ubiquitinated target protein which is rapidlydetected and degraded by the 26S proteasome.

The ubiquitination of these proteins is mediated by a cascade ofenzymatic activity. Ubiquitin is first activated in an ATP-dependentmanner by a ubiquitin activating enzyme (E1). The C-terminus of aubiquitin forms a high energy thiolester bond with E 1. The ubiquitin isthen passed to a ubiquitin conjugating enzyme (E2; also called ubiquitincarrier protein), also linked to this second enzyme via a thiolesterbond. The ubiquitin is finally linked to its target protein to form aterminal isopeptide bond under the guidance of a ubiquitin ligase (E3).In this process, chains of ubiquitin are formed on the target protein,each covalently ligated to the next through the activity of E3.

The components of the ubiquitin ligation cascade have receivedconsiderable attention. E1 and E2 are structurally related and wellcharacterized enzymes. There are several species of E2, some of whichact in preferred pairs with specific E3 enzymes to confer specificityfor different target proteins. E3 enzymes contain two separateactivities: a ubiquitin ligase different target proteins. E3 enzymescontain two separate activities: a ubiquitin ligase activity toconjugate ubiquitin to substrates and form polyubiquitin chains viaisopeptide bonds, and a targeting activity to physically bring theligase and substrate together. Substrate specificity of different E3enzymes is the major determinant in the selectivity of theubiquitin-dependent protein degradation process.

E3 ligases that have been characterized include the HECT (homologous toE6-AP carboxy terminus) domain proteins, represented by the mammalianE6AP-E6 complex which functions as a ubiquitin ligase for the tumorsuppressor p53 and which is activated by papillomavirus in cervicalcancer (Huang et al., Science 286:1321-26 (1999)). The bestcharacterized E3 ligase is the APC (anaphase promoting complex), whichis a multi-subunit complex that is required for both entry into anaphaseas well as exit from mitosis (see King et al., Science 274:1652-59(1996) for review). The APC plays a crucial role in regulating thepassage of cells through anaphase by promoting ubiquitin-dependentproteolysis of many proteins. In addition to degrading the mitoticB-type cyclin for inactivation of CDC2 kinase activity, the APC is alsorequired for degradation of other proteins for sister chromatidseparation and spindle disassambly. Most proteins known to be degradedby the APC contain a conserved nine amino acid motif known as the“destruction box” that targets them for ubiquitination and subsequentdegradation. However, proteins that are degraded during G1, including G1cyclins, CDK inhibitors, transcription factors and signalingintermediates, do not contain this conserved amino acid motif. Instead,substrate phosphorylation appears to play an important role in targetingtheir interaction with an E3 ligase for ubiquitination (see Hershko etal., Ann. Rev. Biochem. 67:429-75 (1998)).

In eukaryotes, a family of complexes with E3 ligase activity play animportant role in regulating G1 progression. These complexes, calledSCF's, consist of at least three subunits, SKP 1, Cullins (having atleast seven family members) and an F-box protein (of which hundreds ofspecies are known) which bind directly to and recruit the substrate tothe E3 complex. The combinatorial interactions between the SCF's and arecently discovered family of RING finger proteins, the ROC/APC11proteins, have been shown to be the key elements conferring ligaseactivity to E3 protein complexes. Particular ROC/Cullin combinations canregulate specific cellular pathways, as exemplified by the function ofAPC 11-APC2, involved in the proteolytic control of sister chromatidseparation and exit from telophase into G1 in mitosis (see King et al.,supra; Koepp et al., Cell 97:431-34 (1999)), and ROC1-Cullin 1, involvedin the proteolytic degradation of I_(κ)B_(α)in NF-_(κ)B/I_(κ)B mediatedtranscription regulation (Tan et al., Mol. Cell 3(4):527-533 (1999);Laney et al., Cell 97:427-30 (1999)).

Because the E3 complex is the major determinant of selection for proteindegradation by the ubiquitin-dependent proteolytic process, modulatorsof E3 ligase activity may be used to upregulate or downregulate specificmolecules involved in cellular signal transduction. Disease processescan be treated by such up- or down regulation of signal transducers toenhance or dampen specific cellular responses. This principle has beenused in the design of a number of therapeutics, includingPhosphodiesterase inhibitors for airway disease and vascularinsufficiency, Kinase inhibitors for malignant transformation andProteasome inhibitors for inflammatory conditions such as arthritis.

Due to the importance of ubiquitination in cellular regulation and thewide array of different possible components in ubiquitin-dependentproteolysis, there is a need for a fast and simple means for assaying E3ligase activity. Furthermore, such an assay would be very useful for theidentification of modulators of E3 ligase. Accordingly, it is an objectof the present invention to provide methods of assaying ubiquitin ligaseactivity, which methods may further be used to identify modulators ofubiquitin ligase activity.

DESCRIPTION OF THE RELATED ART

Tan et al., supra, disclose that ROC1/Cullin catalyzes ubiquitinpolymerization in the absence of target protein substrate. Ohta et al.,Mol. Cell 3(4):535-541 (1999) disclose that APC11/APC2 also catalyzeubiquitin polymerization in the absence of target protein substrate, andthat this activity is dependent on the inclusion of the proper E2species. Rolfe et al., U.S. Pat. No. 5,968,761 disclose an assay foridentifying inhibitors of ubiquitination of a target regulatory protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relative amounts of fluorescently labeled ubiquitin ina ubiquitin activating and conjugating assay. In these experiments, E2is His-Ubch5c.

FIG. 2 shows the relative amounts of ubiquitin ligase activity resultingfrom various combinations of ubiquitination enzymes. In theseexperiments, E3 comprises the RING finger protein ROC1 and the CullinCul1.

FIG. 3 shows relative ubiquitin ligase activity in an assay combiningubiquitin, E1, E2 and E3. FIG. 3A shows relative ubiquitin ligaseactivity using varying amounts of E1 in the presence and absence ofDMSO. FIG. 3B shows relative ubiquitin ligase activity using varyingamounts of ubiquitin and E3.

FIG. 4 shows the signal to noise ratio of fluorescent label in aubiquitin ligase activity assay utilizing FLAG (DYKDDDDK; SEQ IDNO:13)-ubiquitin and an anti-FLAG (DYKDDDDK; SEQ ID NO:13)/anti-mouseantibody conjugated to HRP and Luminol fluorescent HRP substrate. Thesignal was measured from a reaction composition comprising E1, E2 andE3, which E3 specifically bound the reaction receptacle surfacesubstrate. The background was measured as the amount of fluorescencepresent after performing the assay in the absence of E3.

FIG. 5 shows the concentration-dependent effect of two ubiquitin ligaseactivity modulators in assays measuring ubiquitin ligase activity withtwo different E3 enzymes. FIG. 5A shows a concentration-dependentreduction in ubiquitin ligase activity in assays comprising eitherROC1Cul1 or ROC2/Cul5 as the components of the E3 ubiquitin ligase. FIG.5B shows a slightly different pattern of concentration-dependentreduction of ubiquitin ligase activity for another modulator.

FIG. 6 shows the proportions of ubiquitin ligase activity and ubiquitinconjugating activity in the presence and absence of two candidateubiquitin ligase enzyme modulators for combinations of E1, E2 and E3 andcombinations of enzymes E1 and E2. FIG. 6A shows a candidateubiquitination enzyme modulator that affects only E3. FIG. 6B showscandidate ubiquitination enzyme modulator that affects enzymes otherthan E3.

FIG. 7 shows the concentration-dependent effects of two candidateubiquitin ligase modulators on ubiquitin ligase activity and ubiquitinconjugating activity. FIG. 7A shows the results for a candidatemodulator having a concentration-dependent effect on ubiquitin ligaseactivity (E1+E2+E3), but not have on ubiquitin conjugating activity(E1+E2), thus affecting only the E3 ligase. FIG. 7B shows the resultsfor a candidate modulator having a concentration-dependent effect onboth ubiquitin conjugating activity and ubiquitin ligase activity, thusaffecting a component other than the E3 ligase.

FIGS. 8A and 8B (SEQ ID NOS:1-2) show the nucleic acid sequence encodingrabbit E1 and the amino acid sequence of rabbit E1, respectively.

FIGS. 9A and 9B (SEQ ID NOS:3-4) show the nucleic acid sequence encodingthe E2 Ubc5c and the amino acid sequence of the E2 Ubc5c, respectively.

FIG. 10 (SEQ ID NO:5) shows the amino acid sequence of the RING fingerprotein APC11.

FIG. 11 (SEQ ID NO:6) shows the amino acid sequence of the RING fingerprotein ROC1.

FIGS. 12A and 12B (SEQ ID NOS:7-8) show the nucleic acid sequenceencoding the RING finger protein ROC2 and the amino acid sequence ofROC2, respectively.

FIGS. 13A and 13B (SEQ ID NOS:9-10) show the nucleic acid sequenceencoding the Cullin CUL5 and the amino acid sequence of CUL5,respectively.

FIGS. 14A and 14B (SEQ ID NOS:11-12) show the nucleic acid sequenceencoding the Cullin APC2 and the amino acid sequence of APC2,respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods for assaying ubiquitinligase activity. In a broad embodiment, the method provides measuringubiquitin ligase activity directly where the reaction has occurred, thusobviating the need for target proteins and subsequent analysis such asseparating ligated material in an SDS PAGE procedure. This thus allowsmulti-well array analysis and high throughput screening techniques formodulators of ligase activity. In addition, the present methods allowthe use of many different combinations of E3 components and E2/E3combinations, without requiring prior identification of specific targetsubstrates.

In general, the method involves combining ubiquitin and ubiquitinligation enzymes and measuring the amount of ubiquitin ligated to aubiquitination substrate protein. In a preferred embodiment, theubiquitination substrate protein is ubiquitin itself, therefore what ismeasured is poly-ubiquitin chains produced in the ligase reaction.

In a preferred embodiment, no specific target protein is used to measureubiquitin ligase activity. By “target protein” herein is meant a proteinother than ubiquitin to which ubiquitin is ligated by ubiquitinationenzymes. In this embodiment, preferably, the poly-ubiquitin chainsmeasured are bound to E3.

In a preferred embodiment, E3 is attached to the surface of a reactionvessel, such as the well of a multi-well plate. This embodimentfacilitates the separation of ligated ubiquitin from unligatedubiquitin. Means of attaching E3 to the surface of a reaction vessel aredescribed below. This embodiment allows the ubiquitin ligase reactionand detection and measurement of ligated ubiquitin to occur in the samevessel, making the assay useful for high-throughput screeningapplications.

In a preferred embodiment, the ubiquitin is labeled, either directly orindirectly, as further described below, and the amount of label ismeasured. This allows for easy and rapid detection and measurement ofligated ubiquitin, making the assay useful for high-throughput screeningapplications. One of ordinary skill in the art will recognize theapplicability of the present invention to screening for agents whichmodulate ubiquitination enzyme activity.

Accordingly, the present invention provides methods and compositions forassaying ubiquitin ligase activity. By “ubiquitin” herein is meant apolypeptide which is ligated to another polypeptide by ubiquitin ligaseenzymes. The ubiquitin can be from any species of organism, preferably aeukaryotic species. Preferably, the ubiquitin is mammalian. Morepreferably, the ubiquitin is human ubiquitin.

In a preferred embodiment, when ubiquitin is ligated to a targetprotein, that protein is targeted for degradation by the 26S proteasome.

Preferred embodiments of the invention utilize a 76 amino acid humanubiquitin. Other embodiments utilize variants of ubiquitin, as furtherdescribed below.

Also encompassed by “ubiquitin” is naturally occurring alleles andman-made variants of such a 76 amino acid polypeptide. In a preferredembodiment, the ubiquitin has the amino acid sequence of amino acids1-76 of ATCC accession number AAA36789 or AAB06013, incorporated hereinby reference. In a preferred embodiment, variants of ubiquitin have anoverall amino acid sequence identity of preferably greater than about75%, more preferably greater than about 80%, even more preferablygreater than about 85% and most preferably greater than 90% of the aminoacid sequence of amino acids 1-76 of ATCC accession number AAA36789 orAAB06013. In some embodiments the sequence identity will be as high asabout 93 to 95 or 98%.

In another preferred embodiment, a ubiquitin protein has an overallsequence similarity with the amino acid sequence of amino acids 1-76 ofATCC accession number AAA36789 or AAB06013 of greater than about 80%,more preferably greater than about 85%, even more preferably greaterthan about 90% and most preferably greater than 93%. In some embodimentsthe sequence identity will be as high as about 95 to 98 or 99%.

As is known in the art, a number of different programs can be used toidentify whether a protein (or nucleic acid as discussed below) hassequence identity or similarity to a known sequence. Sequence identityand/or similarity is determined using standard techniques known in theart, including, but not limited to, the local sequence identityalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thesequence identity alignment algorithm of Needleman & Wunsch, J. Mol.Biol. 48:443 (1970), by the search for similarity method of Pearson &Lipman, PNAS USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Drive,Madison, Wis.), the Best Fit sequence program described by Devereux etal., Nucl. Acid Res. 12:387-395 (1984), preferably using the defaultsettings, or by inspection. Preferably, percent identity is calculatedby FastDB based upon the following parameters: mismatch penalty of 1;gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30,“Current Methods in Sequence Comparison and Analysis,” MacromoleculeSequencing and Synthesis, Selected Methods and Applications, pp 127-149(1988), Alan R. Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987); the method is similar to that described by Higgins &Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin etal., PNAS USA 90:5873-5787 (1993). A particularly useful BLAST programis the WU-BLAST-2 program which was obtained from Altschul et al.,Methods in Enzymology, 266: 460-480 (1996);http://blast.wustl/edu/blast/README.html]. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al. Nucleic Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions; charges gap lengths of k a cost of 10+k;X_(u) set to 16, and X_(g) set to 40 for database search stage and to 67for the output stage of the algorithms. Gapped alignments are triggeredby a score corresponding to ˜22 bits.

A percent amino acid sequence identity value is determined by the numberof matching identical residues divided by the total number of residuesof the “longer” sequence in the aligned region. The “longer” sequence isthe one having the most actual residues in the aligned region (gapsintroduced by WU-Blast-2 to maximize the alignment score are ignored).

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the amino acid sequence of amino acids 1-76 ofATCC accession number AAA36789 or AABO6013, it is understood that in oneembodiment, the percentage of sequence identity will be determined basedon the number of identical amino acids in relation to the total numberof amino acids. Thus, for example, sequence identity of sequencesshorter than that of amino acids 1-76 of ATCC accession number AAA36789or AAB06013, as discussed below, will be determined using the number ofamino acids in the shorter sequence, in one embodiment. In percentidentity calculations relative weight is not assigned to variousmanifestations of sequence variation, such as, insertions, deletions,substitutions, etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0”,which obviates the need for a weighted scale or parameters as describedbelow for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the “shorter”sequence in the aligned region and multiplying by 100. The “longer”sequence is the one having the most actual residues in the alignedregion.

Ubiquitin proteins of the present invention may be shorter or longerthan the amino acid sequence of amino acids 1-76 of ATCC accessionnumber AAA36789 or AAB06013. Thus, in a preferred embodiment, includedwithin the definition of ubiquitin are portions or fragments of theamino acid sequence of amino acids 1-76 of ATCC accession numberAAA36789 or AAB06013. In one embodiment herein, fragments of ubiquitinare considered ubiquitin proteins if they are ligated to anotherpolypeptide by ubiquitin ligase enzymes.

In addition, as is more fully outlined below, ubiquitin can be madelonger than that depicted in the amino acid sequence of amino acids 1-76of ATCC accession number AAA36789 or AAB06013; for example, by theaddition of tags, the addition of other fusion sequences, or theelucidation of additional coding and non-coding sequences. As describedbelow, the fusion of a ubiquitin peptide to a fluorescent peptide, suchas Green Fluorescent Peptide (GFP), is particularly preferred.

The ubiquitin protein, as well as other proteins of the presentinvention, are preferably recombinant. A “recombinant protein” is aprotein made using recombinant techniques, i.e. through the expressionof a recombinant nucleic acid as described below. In a preferredembodiment, the ubiquitin of the invention is made through theexpression of the nucleic acid of ATCC accession number M26880 orU49869, or fragment thereof. A recombinant protein is distinguished fromnaturally occurring protein by at least one or more characteristics. Forexample, the protein may be isolated or purified away from some or allof the proteins and compounds with which it is normally associated inits wild type host, and thus may be substantially pure. For example, anisolated protein is unaccompanied by at least some of the material withwhich it is normally associated in its natural state, preferablyconstituting at least about 0.5%, more preferably at least about 5% byweight of the total protein in a given sample. A substantially pureprotein comprises at least about 75% by weight of the total protein,with at least about 80% being preferred, and at least about 90% beingparticularly preferred. The definition includes the production of aprotein from one organism in a different organism or host cell.Alternatively, the protein may be made at a significantly higherconcentration than is normally seen, through the use of an induciblepromoter or high expression promoter, such that the protein is made atincreased concentration levels. Alternatively, the protein may be in aform not normally found in nature, as in the addition of an epitope tagor amino acid substitutions, insertions and deletions, as discussedbelow.

As used herein and further defined below, “nucleic acid” may refer toeither DNA or RNA, or molecules which contain both deoxy- andribonucleotides. The nucleic acids include genomic DNA, cDNA andoligonucleotides including sense and anti-sense nucleic acids. Suchnucleic acids may also contain modifications in the ribose-phosphatebackbone to increase stability and half life of such molecules inphysiological environments.

The nucleic acid may be double stranded, single stranded, or containportions of both double stranded or single stranded sequence. As will beappreciated by those in the art, the depiction of a single strand(“Watson”) also defines the sequence of the other strand (“Crick”); thusthe sequences depicted in the figures also include the complement of thesequence. By the term “recombinant nucleic acid” herein is meant nucleicacid, originally formed in vitro, in general, by the manipulation ofnucleic acid by endonucleases, in a form not normally found in nature.Thus an isolated nucleic acid, in alinear form, or an expression vectorformed in vitro by ligating DNA molecules that are not normally joined,are both considered recombinant for the purposes of this invention. Itis understood that once a recombinant nucleic acid is made andreintroduced into a host cell or organism, it will replicatenon-recombinantly, i.e. using the in vivo cellular machinery of the hostcell rather than in vitro manipulations; however, such nucleic acids,once produced recombinantly, although subsequently replicatednon-recombinantly, are still considered recombinant for the purposes ofthe invention.

The terms “polypeptide” and “protein” may be used interchangeablythroughout this application and mean at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides andpeptides. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. Thus “aminoacid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example, homo-phenylalanine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The side chains may be in either the (R) orthe (S) configuration. In the preferred embodiment, the amino acids arein the (S) or L-configuration. If non-naturally occurring side chainsare used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradation.

In one embodiment, the present invention provides compositionscontaining protein variants, for example ubiquitin, E1, E2 and/or E3variants. These variants fall into one or more of three classes:substitutional, insertional or deletional variants. These variantsordinarily are prepared by site specific mutagenesis of nucleotides inthe DNA encoding a protein of the present compositions, using cassetteor PCR mutagenesis or other techniques well known in the art, to produceDNA encoding the variant, and thereafter expressing the DNA inrecombinant cell culture as outlined above. However, variant proteinfragments having up to about 100-150 residues may be prepared by invitro synthesis using established techniques. Amino acid sequencevariants are characterized by the predetermined nature of the variation,a feature that sets them apart from naturally occurring allelic orinterspecies variation of the protein amino acid sequence. The variantstypically exhibit the same qualitative biological activity as thenaturally occurring analogue, although variants can also be selectedwhich have modified characteristics as will be more fully outlinedbelow.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed variants screened for theoptimal desired activity. Techniques for making substitution mutationsat predetermined sites in DNA having a known sequence are well known,for example, M13 primer mutagenesis and PCR mutagenesis. Rapidproduction of many variants may be done using techniques such as themethod of gene shuffling, whereby fragments of similar variants of anucleotide sequence are allowed to recombine to produce new variantcombinations. Examples of such techniques are found in U.S. Pat. Nos.5,605,703; 5,811,238; 5,873,458; 5,830,696; 5,939,250; 5,763,239;5,965,408; and 5,945,325, each of which is incorporated by referenceherein in its entirety. Screening of the mutants is done using ubiquitinligase activity assays of the present invention.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the protein are desired,substitutions are generally made in accordance with the following chart:

CHART 1 Original Residue Exemplary Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser, Ala Gln Asn Glu Asp Gly Pro His Asn, Gln IleLeu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, TyrSer Thr Thr Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inChart I. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g. seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by)an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g. phenylalanine, is substituted for (orby) one not having a side chain, e.g. glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the proteins as needed. Alternatively, the variantmay be designed such that the biological activity of the protein isaltered. For example, glycosylation sites may be altered or removed.

Covalent modifications of polypeptides are included within the scope ofthis invention. One type of covalent modification includes reactingtargeted amino acid residues of a polypeptide with an organicderivatizing agent that is capable of reacting with selected side chainsor the N-or C-terminal residues of a polypeptide. Derivatization withbifunctional agents is useful, for instance, for crosslinking a proteinto a water-insoluble support matrix or surface for use in the method forscreening assays, as is more fully described below. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, -hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of the aminogroups of lysine, arginine, and histidine side chains [T. E. Creighton,Proteins: Structure and Molecular Properties, W.H. Freeman & Co., SanFrancisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, andamidation of any C-terminal carboxyl group.

Another type of covalent modification of a polypeptide included withinthe scope of this invention comprises altering the native glycosylationpattern of the polypeptide. “Altering the native glycosylation pattern”is intended for purposes herein to mean deleting one or morecarbohydrate moieties found in native sequence polypeptide, and/oradding one or more glycosylation sites that are not present in thenative sequence polypeptide.

Addition of glycosylation sites to polypeptides may be accomplished byaltering the amino acid sequence thereof. The alteration may be made,for example, by the addition of, or substitution by, one or more serineor threonine residues to the native sequence polypeptide (for O-linkedglycosylation sites). The amino acid sequence may optionally be alteredthrough changes at the DNA level, particularly by mutating the DNAencoding the polypeptide at preselected bases such that codons aregenerated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on apolypeptide is by chemical or enzymatic coupling of glycosides to thepolypeptide. Such methods are described in the art, e.g., in WO 87/05330published Sep. 11, 1987, and in Aplin and Wriston, CRC Crit. Rev.Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of a protein comprises linking thepolypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

Polypeptides of the present invention may also be modified in a way toform chimeric molecules comprising a first polypeptide fused to another,heterologous polypeptide or amino acid sequence. In one embodiment, sucha chimeric molecule comprises a fusion of a ubiquitin polypeptide (or anE2 or an E3, as defined below) with a tag polypeptide which provides anepitope to which an anti-tag antibody can selectively bind. The epitopetag is generally placed at the amino-or carboxyl-terminus of thepolypeptide. The presence of such epitope-tagged forms of a polypeptidecan be detected using an antibody against the tag polypeptide. Also,provision of the epitope tag enables the polypeptide to be readilypurified by affinity purification using an anti-tag antibody or anothertype of affinity matrix that binds to the epitope tag. In an alternativeembodiment, the chimeric molecule may comprise a fusion of a polypeptidedisclosed herein with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule, such afusion could be to the Fc region of an IgG molecule. Tags for componentsof the invention are defined and described in detail below.

The present invention provides methods for assaying ubiquitin ligaseactivity. By “ubiquitin ligase” is meant a ubiquitination enzyme capableof catalyzing the covalent binding of a ubiquitin to another protein.Preferred embodiments of the invention involve combining ubiquitin andubiquitination enzymes, including ubiquitin ligase, and measuring theamount of ubiquitin (poly-ubiquitin) bound to the ubiquitin ligase. In apreferred embodiment, the ubiquitin ligase is an E3 ubiquitin ligase,defined below.

Embodiments of the present invention involve binding ubiquitin to aubiquitination substrate protein. By “ubiquitination substrate protein”is meant a protein to which the ubiquitin ligase can catalyze thecovalent binding of ubiquitin and includes target proteins andubiquitin. In a preferred embodiment, the ubiquitination substrateprotein is ubiquitin and the ubiquitin ligase catalyzes the formation ofpolyubiquitin chains. Preferably, the polyubiquitin chains are formed bythe ubiquitin ligase in the absence of any target protein.

In a preferred embodiment, the invention is directed to a method ofassaying ubiquitin ligase activity. By “ubiquitin ligase activity”,“ubiquitin ligation” and grammatical equivalents thereof is meant thecatalysis of the covalent binding of ubiquitin to a substrate protein.Preferably, each ubiquitin is bound such that a subsequent ubiquitinpolypeptide may be bound to it, to form chains comprising a plurality ofubiquitin molecules. In a preferred embodiment, ubiquitin ligaseactivity occurs in the absence of target proteins.

In a preferred embodiment, the invention is additionally directed to amethod of assaying ubiquitin activating activity. By “ubiquitinactivating activity”, “ubiquitin activation” and grammatical equivalentsthereof is meant the binding of ubiquitin and E1. Preferably, E1 forms ahigh energy thiolester bond with the ubiquitin.

In a preferred embodiment, the invention is also directed to a method ofassaying ubiquitin conjugating activity. By “ubiquitin conjugatingactivity”, “ubiquitin conjugation” and grammatical equivalents thereofis meant the binding of activated ubiquitin with E2. As will 30 beappreciated by those in the art, due to the presence of the high energythiolester bond in the E2-ubiquitin conjugate, conjugated ubiquitin maybe joined to other ubiquitin at a low rate in the absence of thecatalytic activity of E3. Therefore, some of the ubiquitin measured in aubiquitin conjugating activity assay will be in the form ofpoly-ubiquitin.

The present invention provides methods and compositions comprisingcombining ubiquitin with other components. By “combining” is meant theaddition of the various components into a receptacle under conditionswhereby ubiquitin ligase activity may take place. In a preferredembodiment, the receptacle is a well of a 96 well plate or othercommercially available multiwell plate. In an alternate preferredembodiment, the receptacle is the reaction vessel of a FACS machine.Other receptacles useful in the present invention include, but are notlimited to 384 well plates and 1536 well plates. Still other receptaclesuseful in the present invention will be apparent to the skilled artisan.

The addition of the components may be sequential or in a predeterminedorder or grouping, as long as the conditions amenable to ubiquitinligase activity are obtained. Such conditions are well known in the art,and further guidance is provided below.

In a preferred embodiment, one or more components of the presentinvention comprise a tag. By “tag” is meant an attached molecule ormolecules useful for the identification or isolation of the attachedcomponent. Components having a tag are referred to as “tag-X”, wherein Xis the component. For example, a ubiquitin comprising a tag is referredto herein as “tag-ubiquitin”. Preferably, the tag is covalently bound tothe attached component. When more than one component of a combinationhas a tag, the tags will be numbered for identification, for example“tag1-ubiquitin”. Preferred tags include, but are not limited to, alabel, a partner of a binding pair, and a surface substrate bindingmolecule. As will be evident to the skilled artisan, many molecules mayfind use as more than one type of tag, depending upon how the tag isused.

By “label” is meant a molecule that can be directly (i.e., a primarylabel) or indirectly (i.e., a secondary label) detected; for example alabel can be visualized and/or measured or otherwise identified so thatits presence or absence can be known. As will be appreciated by those inthe art, the manner in which this is done will depend on the label.Preferred labels include, but are not limited to, fluorescent labels,label enzymes and radioisotopes.

By “fluorescent label” is meant any molecule that may be detected viaits inherent fluorescent properties. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, andTexas Red. Suitable optical dyes are described in the 1996 MolecularProbes Handbook by Richard P. Haugland, hereby expressly incorporated byreference. Suitable fluorescent labels also include, but are not limitedto, green fluorescent protein (GFP; Chalfie, et al., Science263(5148):802-805 (Feb. 11, 1994); and EGFP; Clontech—Genbank AccessionNumber U55762 ), blue fluorescent protein (BFP; 1. QuantumBiotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor,Montreal (Quebec) Canada H3H 1J9; 2. Stauber, R. H. Biotechniques24(3):462-471 (1998); 3. Heim, R. and Tsien, R. Y. Curr. Biol. 6:178-182(1996)), enhanced yellow fluorescent protein (EYFP; 1. ClontechLaboratories, Inc., 1020 East Meadow Circle, Palo Alto, Calif. 94303),luciferase (Ichiki, et al., J. Immunol. 150(12):5408-5417 (1993)),β-galactosidase (Nolan, et al., Proc Natl Acad Sci USA 85(8):2603-2607(April 1988)) and Renilla WO 92/15673; WO 95/07463; WO 98/14605; WO98/26277; WO 99/49019; U.S. Pat. No. 5,292,658; U.S. Pat. No. 5,418,155;U.S. Pat. No. 5,683,888; U.S. Pat. No. 5,741,668; U.S. Pat. No.5,777,079; U.S. Pat. No. 5,804,387; U.S. Pat. No. 5,874,304; U.S. Pat.No. 5,876,995; and U.S. Pat. No. 5,925,558) All of the above-citedreferences are expressly incorporated herein by reference.

By “label enzyme” is meant an enzyme which may be reacted in thepresence of a label enzyme substrate which produces a detectableproduct. Suitable label enzymes for use in the present invention includebut are not limited to, horseradish peroxidase, alkaline phosphatase andglucose oxidase. Methods for the use of such substrates are well knownin the art. The presence of the label enzyme is generally revealedthrough the enzyme's catalysis of a reaction with a label enzymesubstrate, producing an identifiable product. Such products may beopaque, such as the reaction of horseradish peroxidase with tetramethylbenzedine, and may have a variety of colors. Other label enzymesubstrates, such as Luminol (available from Pierce Chemical Co.), havebeen developed that produce fluorescent reaction products. Methods foridentifying label enzymes with label enzyme substrates are well known inthe art and many commercial kits are available. Examples and methods forthe use of various label enzymes are described in Savage et al.,Previews 247:6-9 (1998), Young, J. Virol. Methods 24:227-236 (1989),which are each hereby incorporated by reference in their entirety.

By “radioisotope” is meant any radioactive molecule. Suitableradioisotopes for use in the invention include, but are not limited to¹⁴C, ³H, ³²P, ³³P, 35S, ¹²⁵I, and ¹³¹I. The use of radioisotopes aslabels is well known in the art.

In addition, labels may be indirectly detected, that is, the tag is apartner of a binding pair. By “partner of a binding pair” is meant oneof a first and a second moiety, wherein said first and said secondmoiety have a specific binding affinity for each other. Suitable bindingpairs for use in the invention include, but are not limited to,antigens/antibodies (for example, digoxigenin/anti-digoxigenin,dinitrophenyl (DNP)/anti-DNP, dansyl-X-anti-dansyl,Fluorescein/anti-fluorescein, lucifer yellow/anti-lucifer yellow, andrhodamine anti-rhodamine), biotin/avid (or biotin/streptavidin) andcalmodulin binding protein (CBP)/calmodulin. Other suitable bindingpairs include polypeptides such as FLAG (DYKDDDDK; SEQ ID NO:13) [Hoppet al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide[Martin et al., Science, 255:192-194 (1994)]; tubilin epitope peptide[skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad.Sci. USA, 87:6393-6397 (1990)] and the antibodies each thereto.Generally, in a preferred embodiment, the smaller of the binding pairpartners serves as the tag, as steric considerations in ubiquitinligation may be important. As will be appreciated by those in the art,binding pair partners may be used in applications other than forlabeling, as is further described below.

As will be appreciated by those in the art, a partner of one bindingpair may also be a partner of another binding pair. For example, anantigen (first moiety) may bind to a first antibody (second moiety)which may, in turn, be an antigen to a second antibody (third moiety).It will be further appreciated that such a circumstance allows indirectbinding of a first moiety and a third moiety via an intermediary secondmoiety that is a binding pair partner to each.

As will be appreciated by those in the art, a partner of a binding pairmay comprise a label, as described above. It will further be appreciatedthat this allows for a tag to be indirectly labeled upon the binding ofa binding partner comprising a label. Attaching a label to a tag whichis a partner of a binding pair, as just described, is referred to hereinas “indirect labeling”.

By “surface substrate binding molecule” and grammatical equivalentsthereof is meant a molecule have binding affinity for a specific surfacesubstrate, which substrate is generally a member of a binding pairapplied, incorporated or otherwise attached to a surface. Suitablesurface substrate binding molecules and their surface substratesinclude, but are not limited to poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags and Nickel substrate; theGlutathione-S Transferase tag and its antibody substrate (available fromPierce Chemical); the flu HA tag polypeptide and its antibody 12CA5substrate [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; thec-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibody substratesthereto [Evan et al., Molecular and Cellular Biology, 5:3610-3616(1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and itsantibody substrate [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. In general, surface binding substrate molecules useful in thepresent invention include, but are not limited to, polyhistidinestructures (His-tags) that bind nickel substrates, antigens that bind tosurface substrates comprising antibody, haptens that bind to avidinsubstrate (e.g., biotin) and CBP that binds to surface substratecomprising calmodulin.

Production of substrate-embedded antibodies is well known; see Slinkinet al., Bioconj. Chem. 2:342-348 (1991); Torchilin et al., supra;Trubetskoy et al., Bioconj. Chem. 3:323-327 (1992); King et al., CancerRes. 54:6176-6185 (1994); and Wilbur et al., Bioconjugate Chem.5:220-235 (1994) (all of which are hereby expressly incorporated byreference), and attachment of or production of proteins with antigens isdescribed above.

Calmodulin-embedded substrates are commercially available, andproduction of proteins with CBP is described in Simcox et al.,Strategies 8:40-43 (1995), which is hereby incorporated by reference inits entirety.

As will be appreciated by those in the art, tag-components of theinvention can be made in various ways, depending largely upon the formof the tag. Components of the invention and tags are preferably attachedby a covalent bond.

The production of tag-polypeptides by recombinant means when the tag isalso a polypeptide is described below. Production of FLAG (DYKDDDDK; SEQID NO:13)-labeled proteins is well known in the art and kits for suchproduction are commercially available (for example, from Kodak andSigma). Methods for the production and use of FLAG (DYKDDDDK; SEQ IDNO:13)-labeled proteins are found, for example, in Winston et al., Genesand Devel. 13:270-283 (1999), incorporated herein in its entirety, aswell as product handbooks provided with the above-mentioned kits.

Biotinylation of target molecules and substrates is well known, forexample, a large number of biotinylation agents are known, includingamine-reactive and thiol-reactive agents, for the biotinylation ofproteins, nucleic acids, carbohydrates, carboxylic acids; see chapter 4,Molecular Probes Catalog, Haugland, 6th Ed. 1996, hereby incorporated byreference. A biotinylated substrate can be attached to a biotinylatedcomponent via avidin or streptavidin. Similarly, a large number ofhaptenylation reagents are also known (Id.).

Methods for labeling of proteins with radioisotopes are known in theart. For example, such methods are found in Ohta et al., Molec. Cell3:535-541 (1999), which is hereby incorporated by reference in itsentirety.

Production of proteins having His-tags by recombinant means is wellknown, and kits for producing such proteins are commercially available.Such a kit and its use is described in the QIAexpress Handbook fromQuiagen by Joanne Crowe et al., hereby expressly incorporated byreference.

The functionalization of labels with chemically reactive groups such asthiols, amines, carboxyls, etc. is generally known in the art. In apreferred embodiment, the tag is functionalized to facilitate covalentattachment.

The covalent attachment of the tag may be either direct or via a linker.In one embodiment, the linker is a relatively short coupling moiety,that is used to attach the molecules. A coupling moiety may besynthesized directly onto a component of the invention, ubiquitin forexample, and contains at least one functional group to facilitateattachment of the tag. Alternatively, the coupling moiety may have atleast two functional groups, which are used to attach a functionalizedcomponent to a functionalized tag, for example. In an additionalembodiment, the linker is a polymer. In this embodiment, covalentattachment is accomplished either directly, or through the use ofcoupling moieties from the component or tag to the polymer. In apreferred embodiment, the covalent attachment is direct, that is, nolinker is used. In this embodiment, the component preferably contains afunctional group such as a carboxylic acid which is used for directattachment to the functionalized tag. It should be understood that thecomponent and tag may be attached in a variety of ways, including thoselisted above. What is important is that manner of attachment does notsignificantly alter the functionality of the component. For example, intag-ubiquitin, the tag should be attached in such a manner as to allowthe ubiquitin to be covalently bound to other ubiquitin to formpolyubiquitin chains. As will be appreciated by those in the art, theabove description of covalent attachment of a label and ubiquitinapplies equally to the attachment of virtually any two molecules of thepresent disclosure.

In a preferred embodiment, the tag is functionalized to facilitatecovalent attachment, as is generally outlined above. Thus, a widevariety of tags are commercially available which contain functionalgroups, including, but not limited to, isothiocyanate groups, aminogroups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonylhalides, all of which may be used to covalently attach the tag to asecond molecule, as is described herein. The choice of the functionalgroup of the tag will depend on the site of attachment to either alinker, as outlined above or a component of the invention. Thus, forexample, for direct linkage to a carboxylic acid group of a ubiquitin,amino modified or hydrazine modified tags will be used for coupling viacarbodiimide chemistry, for example using1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) as is known in theart (see Set 9 and Set 11 of the Molecular Probes Catalog, supra; seealso the Pierce 1994 Catalog and Handbook, pages T-155 to T-200, both ofwhich are hereby incorporated by reference). In one embodiment, thecarbodiimide is first attached to the tag, such as is commerciallyavailable for many of the tags described herein.

In a preferred embodiment, ubiquitin is in the form of tag-ubiquitin.

In a preferred embodiment, ubiquitin is in the form of tag-ubiquitin,wherein, tag is a partner of a binding pair. Preferably in thisembodiment the tag is FLAG (DYKDDDDK; SEQ ID NO:13) and the bindingpartner is anti-FLAG (DYKDDDDK; SEQ ID NO:13). Preferably in thisembodiment, a label is attached to FLAG (DYKDDDDK; SEQ ID NO:13) byindirect labeling. Preferably,the label is a label enzyme. Mostpreferably, the label enzyme is horseradish peroxidase, which is reactedwith a fluorescent label enzyme substrate. Preferably, the label enzymesubstrate is Luminol. Alternatively, the label is a fluorescent. label.

The present invention provides methods and compositions comprisingcombining ubiquitin and E1. By “E1” is meant a ubiquitin activatingenzyme. In a preferred embodiment, E1 is capable of transferringubiquitin to an E2, defined below. In a preferred embodiment, E1 bindsubiquitin. In a preferred embodiment, E1 forms a high energy thiolesterbond with ubiquitin, thereby “activating” the ubiquitin.

In a preferred embodiment, E1 proteins useful in the invention includethose having the amino acid sequence of the polypeptide having ATCCaccession numbers A38564, S23770, AAA61246, P22314, CAA40296 andBAA33144, incorporated herein by reference. In a preferred embodiment,E1 has the amino acid sequence shown in FIG. 8B (SEQ ID NO:2) or isencoded by a nucleic acid comprising the sequence shown in FIG. 8A (SEQID NO:1). E1 is commercially available from Affiniti Research Products(Exeter, U.K.).

In a preferred embodiment, nucleic acids which may be used for producingE1 proteins for the invention include, but are not limited to, thosedisclosed by ATCC accession numbers M58028, X56976 and AB012190,incorporated herein by reference. In a preferred embodiment, E1 isencoded by a nucleic acid having a sequence consisting essentially ofthe sequence shown in FIG. 8A (SEQ ID NO:1). Variants of the cited E1proteins, also included in the term “E1”, can be made as describedherein

In a preferred embodiment, the compositions of the invention compriseE2. By “E2” is meant a ubiquitin carrier enzyme (also known as aubiquitin conjugating enzyme). In a preferred embodiment, ubiquitin istransferred from E1 to E2. In a preferred embodiment, the transferresults in a thiolester bond formed between E2 and ubiquitin. In apreferred embodiment, E2 is capable of transferring ubiquitin to an E3,defined below. In a preferred embodiment, the ubiquitination substrateprotein is ubiquitin.

In a preferred embodiment, proteins which may be used in the presentinvention as E2 include, but are not limited to, those having the aminoacid sequences disclosed in ATCC accession numbers AAC37534, P49427,CAA82525, AAA58466, AAC41750, P51669, AAA91460, AAA91461, CAA63538,AAC50633, P27924, AAB36017, Q16763, AAB86433, AAC26141, CAA04156,BAA11675, Q16781 and CAB45853, each of which is incorporated herein byreference. In a preferred embodiment, E2 is Ubc5c. In a preferredembodiment, E2 has the amino acid sequence shown in FIG. 9B (SEQ IDNO:4) or is encoded by a nucleic acid consisting essentially of thesequence shown in FIG. 9A (SEQ ID NO:3). Also specifically includedwithin the term “E2” are variants of E2, which can be made as describedherein.

In a preferred embodiment, nucleic acids which may be used to make E2include, but are not limited to, those nucleic acids having sequencesdisclosed in ATCC accession numbers L2205, Z29328, M92670, L40146,U39317, U39318, X92962, U58522, S81003, AF031141, AF075599, AJ000519,and D83004, each of which is incorporated herein by reference. In apreferred embodiment, the nucleic acid used to make E2 comprises thesequence shown in FIG. 9A (SEQ ID NO:3). As described above, variants ofthese and other E2 encoding, nucleic acids may also be used to makevariant E2 proteins.

In a preferred embodiment, E2 has a tag, as defined above, with thecomplex being referred to herein as “tag-E2”. Preferred E2 tags include,but are not limited to, labels, partners of binding pairs and substratebinding elements. In a most preferred embodiment, the tag is a His-tagor GST-tag.

The present invention provides methods and compositions comprising E3.By “E3” is meant a ubiquitin ligase, as defined above, comprising one ormore components associated with ligation of ubiquitin to aubiquitination substrate protein for ubiquitin-dependent proteolysis. Ina preferred embodiment, E3 comprises a ring finger protein and a Cullin.In a preferred embodiment, RING finger proteins include, but are notlimited to, ROC1, ROC2 and APC11. In a preferred embodiment, Cullinsinclude, but are not limited to, CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5and APC2.

In a preferred embodiment, RING finger proteins include, but are notlimited to, proteins having the amino acid sequence disclosed in ATCCaccession numbers AAD30147 and AAD30146 and 6320196, incorporated hereinby reference. In a more preferred embodiment, the ring finger proteinhas a sequence selected from the group consisting of that shown in FIGS.10, 11, and 12B (SEQ ID NOS:5, 6 and 8).

In a preferred embodiment, Cullins include, but are not limited to,proteins having the amino acid sequences disclosed in ATCC accessionnumbers 4503161, AAC50544, AAC36681, 4503163, AAC51190, AAD23581,4503165, AAC36304, AAC36682, AAD45191, AAC50548, Q13620, 4503167 andAAF0575 1, each of which is incorporated herein by reference. In apreferred embodiment, the Cullin has a sequence as shown in FIG. 13B or14B (SEQ ID NO:10 or 12). In addition, in the context of the invention,each of the RING finger proteins and Cullins encompass variants of theknown or listed sequences, as described herein.

These E3 proteins and variants may be made as described herein. In apreferred embodiment, nucleic acids used to make the RING fingerproteins include, but are not limited to, those having the nucleic acidsequences disclosed in ATCC accession numbers AF142059, AF142060 andnucleic acids 433493 to 433990 of NC 001136. In a preferred embodiment,Cullins are made from nucleic acids including, but not limited to, thosehaving nucleic acid sequences disclosed in ATCC accession numbers NM003592, U58087, AF062536, AF126404, NM 003591, U83410, NM 003590,AB014517, AF062537, AF064087, AF077188, U58091, NM 003478, X81882 andAF191337, each of which is incorporated herein by reference. Asdescribed above, variants of these sequences are also encompassed by theinvention.

In an alternate embodiment, E3 comprises the ligase E3-alpha, E3A(E6-AP), HERC2, SMURF1 or NEDD-4. In this embodiment, the ligase has theamino acid sequence of that disclosed in ATCC accession number AAC39845,Q05086, CAA66655, CAA66654, CAA66656, AAD08657, AAF08298 or P46934, eachof which is incorporated herein by reference. As above, variants arealso encompassed by the invention. Nucleic acids for making E3 for thisembodiment include, but are not limited to, those having the sequencesdisclosed in ATCC accession numbers AF061556, X898032, X9803 1, X98033,AF071172, AF199364 and D42055 and variants thereof.

E3 may also comprise other components, such as SKP1 and F-box proteins.The amino acid and nucleic acid sequences for SKP1 are found in ATCCaccession numbers AAC50241 and U33760, respectively. Many F-box proteinsare known in the art and their amino acid and nucleic acid sequences arereadily obtained by the skilled artisan from various published sources.

In a preferred embodiment, the E3 components are produced recombinantly,as described herein.

In a preferred embodiment, the E3 components are co-expressed in thesame host cell. Co-expression may be achieved by transforming the cellwith a vector comprising nucleic acids encoding two or more of the E3components, or by transforming the host cell with separate vectors, eachcomprising a single component of the desired E3 protein complex. In apreferred embodiment, the RING finger protein and Cullin are expressedin a single host transfected with two vectors, each comprising nucleicacid encoding one or the other polypeptide, as described in furtherdetail in the Examples.

In a preferred embodiment, E3 has a tag, which complex is referred toherein as “tag-E3”. Preferably, the tag is attached to only onecomponent of the E3. Preferred E3 tags include, but are not limited to,labels, partners of binding pairs and substrate binding elements. Morepreferably, the tag is a surface substrate binding molecule. Mostpreferably, the tag is a His-tag or GST-tag.

In an embodiment herein, ubiquitin and ubiquitination enzymes and theircomponents are cloned and expressed as outlined below. Thus, probe ordegenerate polymerase chain reaction (PCR) primer sequences may be usedto find other related ubiquitination proteins from humans or otherorganisms. As will be appreciated by those in the art, particularlyuseful probe and/or PCR primer sequences include the unique areas of anucleic acid sequence. As is generally known in the art, preferred PCRprimers are from about 15 to about 35 nucleotides in length, with fromabout 20 to about 30 being preferred, and may contain inosine as needed.The conditions for the PCR reaction are well known in the art. It istherefore also understood that provided along with the sequences in thesequences cited herein are portions of those sequences, wherein uniqueportions of 15 nucleotides or more are particularly preferred. Theskilled artisan can routinely synthesize or cut a nucleotide sequence tothe desired length.

Once isolated from its natural source, e.g., contained within a plasmidor other vector or excised therefrom as a linear nucleic acid segment,the recombinant nucleic acid can be further-used as a probe to identifyand isolate other nucleic acids. It can also be used as a “precursor”nucleic acid to make modified or variant nucleic acids and proteins.

Using the nucleic acids of the present invention which encode a protein,a variety of expression vectors are made. The expression vectors may beeither self-replicating extrachromosomal vectors or vectors whichintegrate into a host genome. Generally, these expression vectorsinclude transcriptional and translational regulatory nucleic acidoperably linked to the nucleic acid encoding the protein. The term“control sequences” refers to DNA sequences necessary for the expressionof an operably linked coding sequence in a particular host organism. Thecontrol sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. As another example, operablylinked refers to DNA sequences linked so as to be contiguous, and, inthe case of a secretory leader, contiguous and in reading fram. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice. The transcriptional and translationalregulatory nucleic acid will generally be appropriate to the host cellused to express the protein; for example, transcriptional andtranslational regulatory nucleic acid sequences from Bacillus arepreferably used to express the protein in Bacillus. Numerous types ofappropriate expression vectors, and suitable regulatory sequences areknown in the art for a variety of host cells.

In general, the transcriptional and translational regulatory sequencesmay include, but are not limited to, promoter sequences, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, and enhancer or activator sequences. In apreferred embodiment, the regulatory sequences include a promoter andtranscriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters.The promoters may be either naturally occurring promoters or hybridpromoters. Hybrid promoters, which combine elements of more than onepromoter, are also known in the art, and are useful in the presentinvention.

In addition, the expression vector may comprise additional elements. Forexample, the expression vector may have two replication systems, thusallowing it to be maintained in two organisms, for example in mammalianor insect cells for expression and in a procaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector contains at least one sequence homologous to the hostcell genome, and preferably two homologous sequences which flank theexpression construct. The integrating vector may be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

In addition, in a preferred embodiment, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selection genes are well known in the art and will vary with the hostcell used.

A preferred expression vector system is a retroviral vector system suchas is generally described in PCT/US97/01019 and PCT/US97/01048, both ofwhich are hereby expressly incorporated by reference.

Proteins of the present invention are produced by culturing a host celltransformed with an expression vector containing nucleic acid encodingthe protein, under the appropriate conditions to induce or causeexpression of the protein. The conditions appropriate for proteinexpression will vary with the choice of the expression vector and thehost cell, and will be easily ascertained by one skilled in the artthrough routine experimentation. For example, the use of constitutivepromoters in the expression vector will require optimizing the growthand proliferation of the host cell, while the use of an induciblepromoter requires the appropriate growth conditions for induction. Inaddition, in some embodiments, the timing of the harvest is important.For example, the baculoviral systems used in insect cell expression arelytic viruses, and thus harvest time selection can be crucial forproduct yield.

Appropriate host cells include yeast, bacteria, archaebacteria, fungi,and insect and animal cells, including mammalian cells. Of particularinterest are Drosophila melanogaster cells, Saccharomyces cerevisiae andother yeasts, E. coli, Bacillus subtilis, SF9 cells, C129 cells, Saos-2cells, Hi-5 cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells.Of greatest interest are E. coli, SF9 cells and Hi-5 cells.

In a preferred embodiment, the proteins are expressed in mammaliancells. Mammalian expression systems are also known in the art, andinclude retroviral systems. A mammalian promoter is any DNA sequencecapable of binding mammalian RNA polymerase and initiating thedownstream (3′) transcription of a coding sequence for a protein intomRNA. A promoter will have a transcription initiating region, which isusually placed proximal to the 5′ end of the coding sequence, and a TATAbox, using a located 25-30 base pairs upstream of the transcriptioninitiation site. The TATA box is thought to direct RNA polymerase II tobegin RNA synthesis at the correct site. A mammalian promoter will alsocontain an upstream promoter element (enhancer element), typicallylocated within 100 to 200 base pairs upstream of the TATA box. Anupstream promoter element determines the rate at which transcription isinitiated and can act in either orientation. Of particular use asmammalian promoters are the promoters from mammalian viral genes, sincethe viral genes are often highly expressed and have a broad host range.Examples include the SV40 early promoter, mouse mammary tumor virus LTRpromoter, adenovirus major late promoter, herpes simplex virus promoter,and the CMV promoter.

Typically, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-translational cleavage and polyadenylation.Examples of transcription terminator and polyadenylation signals includethose derived form SV40.

The methods of introducing exogenous nucleic acid into mammalian hosts,as well as other hosts, is well known in the art, and will vary with thehost cell used. Techniques include dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, viral infection, encapsulation ofthe polynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

In a preferred embodiment, proteins are expressed in bacterial systems.Bacterial expression systems are well known in the art.

A suitable bacterial promoter is any nucleic acid sequence capable ofbinding bacterial RNA polymerase and initiating the downstream (3′)transcription of the coding sequence of a protein into mRNA. A bacterialpromoter has a transcription initiation region which is usually placedproximal to the 5′ end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site anda transcription initiation site. Sequences encoding metabolic pathwayenzymes provide particularly useful promoter sequences. Examples includepromoter sequences derived from sugar metabolizing enzymes, such asgalactose, lactose and maltose, and sequences derived from biosyntheticenzymes such as tryptophan. Promoters from bacteriophage may also beused and are known in the art. In addition, synthetic promoters andhybrid promoters are also useful; for example, the tac promoter is ahybrid of the trp and lac promoter sequences. Furthermore, a bacterialpromoter can include naturally occurring promoters of non-bacterialorigin that have the ability to bind bacterial RNA polymerase andinitiate transcription.

In addition to a functioning promoter sequence, an efficient ribosomebinding site is desirable. In E. Coli, the ribosome binding site iscalled the Shine-Delgamo (SD) sequence and includes an initiation codonand a sequence 3-9 nucleotides in length located 3-11 nucleotidesupstream of the initiation codon.

The expression vector may also include a signal peptide sequence thatprovides for secretion of the protein in bacteria. The signal sequencetypically encodes a signal peptide comprised of hydrophobic amino acidswhich direct the secretion of the protein from the cell, as is wellknown in the art. The protein is either secreted into the growth media(gram-positive bacteria) or into the periplasmic space, located betweenthe inner and outer membrane of the cell (gram-negative bacteria).

The bacterial expression vector may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed. Suitable selection genes include genes which render thebacteria resistant to drugs such as ampicillin, chloramphenicol,erythromycin, kanamycin, neomycin and tetracycline. Selectable markersalso include biosynthetic genes, such as those in the histidine,tryptophan and leucine biosynthetic pathways.

These components are assembled into expression vectors. Expressionvectors for bacteria are well known in the art, and include vectors forBacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcuslividans, among others.

The bacterial expression vectors are transformed into bacterial hostcells using techniques well known in the art, such as calcium chloridetreatment, electroporation, and others.

In one embodiment, proteins are produced in insect cells. Expressionvectors for the transformation of insect cells, and in particular,baculovirus-based expression vectors, are well known in the art.

In a preferred embodiment, proteins are produced in yeast cells. Yeastexpression systems are well known in the art, and include expressionvectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa,Hansenula polymorpha, Kluyveromyces fragilis and K lactis, Pichiaguillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowialipolytica. Preferred promoter sequences for expression in yeast includethe inducible GAL1,10 promoter, the promoters from alcoholdehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase,glyceraldehyde-3-phosphate-dehydrogenase, hexokinase,phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and theacid phosphatase gene. Yeast selectable markers include ADE2, HIS4,LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; theneomycin phosphotransferase gene, which confers resistance to G418; andthe CUP1 gene, which allows yeast to grow in the presence of copperions.

The protein may also be made as a fusion protein, using techniques wellknown in the art. Thus, for example, the protein may be made as a fusionprotein to increase expression, or for other reasons. For example, whenthe protein is a peptide, the nucleic acid encoding the peptide may belinked to other nucleic acid for expression purposes. Similarly,proteins of the invention can be linked to protein labels, such as greenfluorescent protein (GFP), red fluorescent protein (RFP), bluefluorescent protein (BFP), yellow fluorescent protein (YFP), etc.

In a preferred embodiment, the protein is purified or isolated afterexpression. Proteins may be isolated or purified in a variety of waysknown to those skilled in the art depending on what other components arepresent in the sample. Standard purification methods includeelectrophoretic, molecular, immunological and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, and chromatofocusing. For example,the ubiquitin protein may be purified using a standard anti-ubiquitinantibody column. Ultrafiltration and diafiltration techniques, inconjunction with protein concentration, are also useful. For generalguidance in suitable purification techniques, see Scopes, R., ProteinPurification, Springer-Verlag, N.Y. (1982). The degree of purificationnecessary will vary depending on the use of the protein. In someinstances no purification will be necessary.

Once made, the compositions find use in a number of applications,including, but not limited to, screens for modulators of ubiquitinligase activity. By “modulator” is meant a compound which can increaseor decrease ubiquitin ligase activity. By “candidate”, “candidateagent”, “candidate modulator”, “candidate ubiquitin ligase activitymodulator” or grammatical equivalents herein is meant any molecule, e.g.proteins (which herein includes proteins, polypeptides, and peptides),small organic or inorganic molecules, polysaccharides, polynucleotides,etc. which are to be tested for ubiquitin ligase activity modulatoractivity. Candidate agents encompass numerous chemical classes. In apreferred embodiment, the candidate agents are organic molecules,particularly small organic molecules, comprising functional groupsnecessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups. The candidate agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more chemical functional groups.

Candidate modulators are obtained from a wide variety of sources, aswill be appreciated by those in the art, including libraries ofsynthetic or natural compounds. As will be appreciated by those in theart, the present invention provides a rapid and easy method forscreening any library of candidate modulators, including the widevariety of known combinatorial chemistry-type libraries.

In a preferred embodiment, candidate modulators are synthetic compounds.Any number of techniques are available for the random and directedsynthesis of a wide variety of organic compounds and biomolecules,including expression of randomized oligonucleotides. See for example WO94/24314, hereby expressly incorporated by reference, which discussesmethods for generating new compounds, including random chemistry methodsas well as enzymatic methods. As described in WO 94/24314, one of theadvantages of the present method is that it is not necessary tocharacterize the candidate modulator prior to the assay; only candidatemodulators that increase or decease ubiquitin ligase activity need beidentified. In addition, as is known in the art, coding tags using splitsynthesis reactions may be done, to essentially identify the chemicalmoieties tested.

Alternatively, a preferred embodiment utilizes libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsthat are available or readily produced.

Additionally, natural or synthetically produced libraries and compoundsare readily modified through conventional chemical, physical andbiochemical means. Known pharmacological agents may be subjected todirected or random chemical modifications, including enzymaticmodifications, to produce structural analogs.

In a preferred embodiment, candidate modulators include proteins,nucleic acids, and chemical moieties.

In a preferred embodiment, the candidate modulator are proteins, asdefined above. In a preferred embodiment, the candidate modulators arenaturally occurring proteins or fragments of naturally occurringproteins. Thus, for example, cellular extracts containing proteins, orrandom or directed digests of proteinaceous cellular extracts, may betested, as is more fully described below. In this way libraries ofprocaryotic and eucaryotic proteins may be made for screening againstany number of ubiquitin ligase compositions. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred.

In a preferred embodiment, the candidate modulators are peptides of fromabout 2 to about 50 amino acids, with from about 5 to about 30 aminoacids being preferred, and from about 8 to about 20 being particularlypreferred. The peptides may be digests of naturally occurring proteinsas is outlined above, random peptides, or “biased” random peptides. By“randomized” or grammatical equivalents herein is meant that eachnucleic acid and peptide consists of essentially random nucleotides andamino acids, respectively. Since generally these random peptides (ornucleic acids, discussed below) are chemically synthesized, they mayincorporate any nucleotide or amino acid at any position. The syntheticprocess can be designed to generate randomized proteins or nucleicacids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized candidate bioactive proteinaceous agents.

The library should provide a sufficiently structurally diversepopulation of randomized agents to effect a probabilistically sufficientrange of diversity to allow interaction with a particular ubiquitinligase enzyme. Accordingly, an interaction library must be large enoughso that at least one of its members will have a structure that interactswith a ubiquitin ligase enzyme. Although it is difficult to gauge therequired absolute size of an interaction library, nature provides a hintwith the immune response: a diversity of 10⁷-10⁸ different antibodiesprovides at least one combination with sufficient affinity to interactwith most potential antigens faced by an organism. Published in vitroselection techniques have also shown that a library size of 10⁷ to 10⁸is sufficient to find structures with affinity for a target. A libraryof all combinations of a peptide 7 to 20 amino acids in length, such asgenerally proposed herein, has the potential to code for 20⁷ (10⁹) to20²⁰. Thus, with libraries of 10⁷ to 10⁸ different molecules the presentmethods allow a “working” subset of a theoretically complete interactionlibrary for 7 amino acids, and a subset of shapes for the 20²⁰ library.Thus, in a preferred embodiment, at least 10⁶, preferably at least 10⁷,more preferably at least 10⁸ and most preferably at least 10⁹ differentsequences are simultaneously analyzed in the subject methods. Preferredmethods maximize library size and diversity.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation ofcysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In a preferred embodiment, the bias is towards peptides or nucleic acidsthat interact with known classes of molecules. For example, when thecandidate modulator is a peptide, it is known that much of intracellularsignaling is carried out via short regions of polypeptides interactingwith other polypeptides through small peptide domains. For instance, ashort region from the HIV-1 envelope cytoplasmic domain has beenpreviously shown to block the action of cellular calmodulin. Regions ofthe Fas cytoplasmic domain, which shows homology to the mastoparan toxinfrom Wasps, can be limited to a short peptide region with death-inducingapoptotic or G protein inducing functions. Magainin, a natural peptidederived from Xenopus, can have potent anti-tumor and anti-microbialactivity. Short peptide fragments of a protein kinase C isozyme (BPKC),have been shown to block nuclear translocation of BPKC in Xenopusoocytes following stimulation. And, short SH-3 target peptides have beenused as psuedosubstrates for specific binding to SH-3 proteins. This isof course a short list of available peptides with biological activity,as the literature is dense in this area. Thus, there is much precedentfor the potential of small peptides to have activity on intracellularsignaling cascades. In addition, agonists and antagonists of any numberof molecules may be used as the basis of biased randomization ofcandidate modulators as well. Thus, a number of molecules or proteindomains are suitable as starting points for the generation of biasedrandomized candidate modulators. A large number of small moleculedomains are known, that confer a common function, structure or affinity.In addition, as is appreciated in the art, areas of weak amino acidhomology may have strong structural homology. A number of thesemolecules, domains, and/or corresponding consensus sequences, are known,including, but are not limited to, SH-2 domains, SH-3 domains,Pleckstrin, death domains, protease cleavage/recognition sites, enzymeinhibitors, enzyme substrates, Traf, etc.

In a preferred embodiment, the candidate modulators are nucleic acids.With reference to candidate modulators, by “nucleic acid” or“oligonucleotide” or grammatical equivalents herein means at least twonucleotides covalently linked together. A nucleic acid of the presentinvention will generally contain phosphodiester bonds, although in somecases, as outlined below, nucleic acid analogs are included that mayhave alternate backbones, comprising, for example, phosphoramide(Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein;Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J.Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487(1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am.Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:14191986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages (seeEgholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed.Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al.,Nature 380:207 (1996), all of which are incorporated by reference).Other analog nucleic acids include those with positive backbones (Denpcyet al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger et al., J. Am. Chem. Soc. 1 10:4470 (1988); Letsingeret al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp169-176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of additional moieties such as labels, or to increase thestability and half-life of such molecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made.Alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

Particularly preferred are peptide nucleic acids (PNA) which includespeptide nucleic acid analogs. These backbones are substantiallynon-ionic under neutral conditions, in contrast to the highly chargedphosphodiester backbone of naturally occurring nucleic acids.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. As used herein, the term“nucleoside” includes nucleotides and nucleoside and nucleotide analogs,and modified nucleosides such as amino modified nucleosides. Inaddition, “nucleoside” includes non-naturally occurring analogstructures. Thus for example the individual units of a peptide nucleicacid, each containing a base, are referred to herein as a nucleoside.

As described above generally for proteins, nucleic acid candidatemodulator may be naturally occurring nucleic acids, random nucleicacids, or “biased” random nucleic acids. For example, digests ofprocaryotic or eucaryotic genomes may be used as is outlined above forproteins. Where the ultimate expression product is a nucleic acid, atleast 10, preferably at least 12, more preferably at least 15, mostpreferably at least 21 nucleotide positions need to be randomized, withmore preferable if the randomization is less than perfect. Similarly, atleast 5, preferably at least 6, more preferably at least 7 amino acidpositions need to be randomized; again, more are preferable if therandomization is less than perfect.

In a preferred embodiment, the candidate modulators are organicmoieties. In this embodiment, as is generally described in WO 94/24314,candidate agents are synthesized from a series of substrates that can bechemically modified. “Chemically modified” herein includes traditionalchemical reactions as well as enzymatic reactions. These substratesgenerally include, but are not limited to, alkyl groups (includingalkanes, alkenes, alkynes and heteroalkyl), aryl groups (includingarenes and heteroaryl), alcohols, ethers, amines, aldehydes, ketones,acids, esters, amides, cyclic compounds, heterocyclic compounds(including purines, pyrimidines, benzodiazepins, beta-lactams,tetracylines, cephalosporins, and carbohydrates), steroids (includingestrogens, androgens, cortisone, ecodysone, etc.), alkaloids (includingergots, vinca, curare, pyrollizdine, and mitomycines), organometalliccompounds, hetero-atom bearing compounds, amino acids, and nucleosides.Chemical (including enzymatic) reactions may be done on the moieties toform new substrates or candidate agents which can then be tested usingthe present invention.

As will be appreciated by those in the art, it is possible to screenmore than one type of candidate modulator at a time. Thus, the libraryof candidate modulators used may include only one type of agent (i.e.peptides), or multiple types (peptides and organic agents). The assay ofseveral candidates at one time is further discussed below.

The present invention provides methods and compositions comprisingcombining several components. In a preferred embodiment, a preferredcombination is tag-ubiquitin, E1, E2, and E3. Preferably the tag is alabel, a partner of a binding pair, or a substrate binding molecule.More preferably, the tag is a fluorescent label or a binding pairpartner. In a preferred embodiment, the tag is a binding pair partnerand the ubiquitin is labeled by indirect labeling. In the indirectlabeling embodiment, preferably the label is a fluorescent label or alabel enzyme. In an embodiment comprising a label enzyme, preferably thesubstrate for that enzyme produces a fluorescent product. In a preferredembodiment, the label enzyme substrate is luminol.

In a preferred embodiment, a preferred combination is tag1-ubiquitin,E1, E2 and tag2-E3. Preferably, tag1 is a label, a partner of a bindingpair, or a substrate binding molecule and tag2 is a different label,partner of a binding pair, or substrate binding molecule. Morepreferably, tag1 is a fluorescent label or a member of a binding pair.When tag1 is a member of a binding pair, preferably tag1 is indirectlylabeled. Still more preferably, tag-1 is indirectly labeled with a labelenzyme. Preferably the label enzyme substrate used to reveal thepresence of the enzyme produces a fluorescent product, and morepreferably is luminol. In the presently described combination,preferably tag2 is a surface substrate binding element, more preferablya His-tag.

In a preferred embodiment, a preferred combination is tag1-ubiquitin, E1and tag2-E2. In this embodiment, preferably; tag1 is a label, a partnerof a binding pair, or a substrate binding molecule and tag2 is adifferent label, partner of a binding pair, or substrate bindingmolecule. More preferably, tag-1 is a label or a member of a bindingpair. When tag1 is a member of a binding pair, preferably tag1 isindirectly labeled. In a preferred embodiment, the tag1 label (direct orindirect) is a fluorescent label or a label enzyme. When the tag1 label(direct or indirect) is a label enzyme, preferably the reactionsubstrate used to reveal the presence of the enzyme produces afluorescent product, and more preferably is luminol. In the presentlydescribed combination, preferably tag2 is a substrate binding element,more preferably a His-tag.

In a preferred embodiment, the compositions of the invention do notcomprise a target protein. In this embodiment, ubiquitin is the soleubiquitination substrate, as discussed above. This differentiates thepresent assays from all previous ubiquitination enzyme assays, whichrequired addition of a target protein as part of the composition.Because the different combinations of E3 and E2 and combinations of E3subunits are specific to particular target proteins, the present assaysare much more versatile, allowing any variation of such combinationswithout first identifying the specific target protein to which thecombination is directed.

The components of the present compositions may be combined in varyingamounts. In a preferred embodiment, ubiquitin is combined at a finalconcentration of from 20 to 200 ng per 100 μl reaction solution, mostpreferable at about 100 ng per 100 μl reaction solution.

In a preferred embodiment, E1 is combined at a final concentration offrom 1 to 50 ng per 100 μl reaction solution, more preferably from 1 ngto 20 ng per 100 μl reaction solution, most preferably from 5 ng to 10ng per 100 μl reaction solution.

In a preferred embodiment, E2 is combined at a final concentration of 10to 100 ng per 100 μl reaction solution, more preferably 10-50 ng per 100μl reaction solution.

In a preferred embodiment, E3 is combined at a final concentration offrom 1 ng to 500 ng per 100 μl reaction solution, more preferably from50 to 400 ng per 100 μl reaction solution, still more preferably from100 to 300 ng per 100 μl reaction solution, most preferably about 100 ngper 100 μl reaction solution.

The components of the invention are combined under reaction conditionsthat favor ubiquitin ligase activity. Generally, this will bephysiological conditions. Incubations may be performed at anytemperature which facilitates optimal activity, typically between 4 and40° C. Incubation periods are selected for optimum activity, but mayalso be optimized to facilitate rapid high through put screening.Typically between 0.5 and 1.5 hours will be sufficient.

A variety of other reagents may be included in the compositions. Theseinclude reagents like salts, solvents, buffers, neutral proteins, e.g.albumin, detergents, etc. which may be used to facilitate optimalubiquitination enzyme activity and/or reduce non-specific or backgroundinteractions. Also reagents that otherwise improve the efficiency of theassay, such as protease inhibitors, nuclease inhibitors, anti-microbialagents, etc., may be used. The compositions will also preferably includeadenosine tri-phosphate (ATP).

The mixture of components may be added in any order that promotesubiquitin ligase activity or optimizes identification of candidatemodulator effects. In a preferred embodiment, ubiquitin is provided in areaction buffer solution, followed by addition of the ubiquitinationenzymes. In an alternate preferred embodiment, ubiquitin is provided ina reaction buffer solution, a candidate modulator is then added,followed by addition of the ubiquitination enzymes.

Once combined, preferred methods of the invention comprise measuring theamount of ubiquitin bound to E3. In an alternate preferred embodiment inwhich the combination lacks E3, preferred methods of the inventioncomprise measuring the amount of ubiquitin bound to E2. As will beunderstood by one of ordinary skill in the art, the mode of measuringwill depend on the specific tag attached to the ubiquitin. As will alsobe apparent to the skilled artisan, the amount of ubiquitin bound willencompass not only the particular ubiquitin protein bound directly tothe ubiquitination enzyme, but also the ubiquitin proteins bound to theformer in a polyubiquitin chain.

In a preferred embodiment, the tag attached to the ubiquitin is afluorescent label. In a preferred embodiment, the tag attached toubiquitin is an enzyme label or a binding pair member which isindirectly labeled with an enzyme label. In this latter preferredembodiment, the enzyme label substrate produces a fluorescent reactionproduct. In these preferred embodiments, the amount of ubiquitin boundis measured by luminescence. Equipment for such measurement iscommercially available and easily used by one of ordinary skill in theart to make such a measurement.

Other modes of measuring bound ubiquitin are well known in the art andeasily identified by the skilled artisan for each of the labelsdescribed herein. For instance, radioisotope labeling may be measured byscintillation counting, or by densitometry after exposure to aphotographic emulsion, or by using a device such as a Phosphorlmager.Likewise, densitometry may be used to measure bound ubiquitin followinga reaction with an enzyme label substrate that produces an opaqueproduct when an enzyme label is used.

In preferred methods of the present invention, E3 is bound to a surfacesubstrate. This may be done directly , as described above for thebinding of a label to ubiquitin. This may also be accomplished usingtag-E3, wherein the tag is a surface substrate binding molecule.

In another preferred embodiment of the invention, E2 is bound to asurface substrate in the absence of E3. This may be done directly, asdescribed above for the binding of a label to ubiquitin. This may alsobe accomplished using tag-E2, wherein the tag is a surface substratebinding molecule.

In the two preferred embodiments described immediately above, E3 and E2are in the form of tag-E3 and tag-E2, respectively, and are bound to asurface substrate via a surface substrate binding molecule tag. Ingeneral, any substrate binding molecule can be used. In a preferredembodiment, the tag is a His-tag and the surface substrate is nickel. Ina preferred embodiment, the nickel surface substrate is present on thesurface of the wells of a multi-well plate, such as a 96 well plate.Such multi-well plates are commercially available. The binding of theenzyme to a surface substrate facilitates the separation of boundubiquitin from unbound ubiquitin. In the present embodiment, the unboundubiquitin is easily washed from the receptacle following the ligationreaction. As will be appreciated by those of skill in the art, the useof any surface substrate binding element and receptacle having thesurface substrate to which it binds will be effective for facilitatingthe separation of bound and unbound ubiquitin.

In an alternative embodiment, E3 or E2 is bound, directly or via asubstrate binding element, to a bead. Following ligation, the beads maybe separated from the unbound ubiquitin and the bound ubiquitinmeasured. In a preferred embodiment, E3 or E2 is bound to beads and thecomposition used includes tag-ubiquitin wherein tag is a fluorescentlabel. In this embodiment, the beads with bound ubiquitin may beseparated using a fluorescence-activated cell sorting (FACS) machine.Methods for such use are described in U.S. patent application Ser. No.09/047,119, which is hereby incorporated in its entirety. The amount ofbound ubiquitin can then be measured.

In a preferred embodiment, the compositions of the invention are used toidentify ubiquitin ligase activity modulators. In this embodiment, thecomposition includes a candidate modulator. In a preferred embodiment,the measured amount of tag-ubiquitin bound to E3 is compared with theamount bound when the candidate modulator is absent from thecomposition, whereby the presence or absence of the modulators effect onubiquitin ligase activity is determined. In this embodiment, whether themodulator enhances or inhibits ubiquitin ligase activity is alsodetermined.

In a preferred embodiment, the composition of the invention containing acandidate modulator lacks E3 and the amount of ubiquitin bound to E2 ismeasured. This embodiment may also comprise the step of comparing theamount of ubiquitin bound to E2 in a composition lacking both E3 and thecandidate modulator, whereby the modulatory activity of the candidate onubiquitination enzymes other than E3 is determined. In a preferredembodiment, the percentage difference in the amount of ubiquitin boundto E2 in the presence and absence of the candidate modulator is comparedwith the percentage difference in the amount bound to E3 in the presenceand absence of candidate modulator, whereby the point of effect of thecandidate modulator in the enzyme cascade is determined. That is, it isdetermined whether the candidate modulator affects E3 ubiquitin ligaseactivity or it affects E1 ubiquitin activating activity and/or E2ubiquitin conjugating activity.

In a preferred embodiment, multiple assays are performed simultaneouslyin a high throughput screening system. In this embodiment, multipleassays may be performed in multiple receptacles, such as the wells of a96 well plate or other multi-well plate. As will be appreciated by oneof skill in the art, such a system may be applied to the assay ofmultiple candidate modulators and/or multiple combination of E3components and/or E2-E3 pairings. In a preferred embodiment, the presentinvention is used in a high-throughput screening system for determiningthe ubiquitin ligase activity of different E2-E3 pairings and/ordifferent E3 component combinations. In an alternate preferredembodiment, the present invention is used in a high throughput screeningsystem for simultaneously testing the effect of individual candidatemodulators.

It is understood by the skilled artisan that the steps of the assaysprovided herein can vary in order. It is also understood, however, thatwhile various options (of compounds, properties selected or order ofsteps) are provided herein, the options are also each providedindividually, and can each be individually segregated from the otheroptions provided herein. Moreover, steps which are obvious and known inthe art that will increase the sensitivity of the assay are intended tobe within the scope of this invention. For example, there may beadditionally washing steps, blocking steps, etc.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are expressly incorporated by reference intheir entirety.

EXAMPLES Example 1 Production of E2, E3 and Ubiquitin

E2 Production

The open reading frame of E2 (Ubc5C) was amplified by PCR and clonedinto the pGex-6p-1 E.Coli. expression vector (Amersham Pharmacia) asBglII-EcoRI fragments, with N-termninus in frame fused to the GST-tag.

Materials and Methods

Plasmid is transformed in BL21 DE3 competent E.coli (Stratagene, cat#230132). Cells are grown at 37° C. in TB+100 ug/ml ampicillin and 0.4%glucose to an OD600 of about 0.6, induced with addition of 320 uM IPTGand allowed to grow for another 3 h before harvest. The pellets arewashed once with cold PBS, then resuspended in about 6 volumes of lysisbuffer (20 rnM Tris, 10% glycerol, 0.5 M Nacl, 2.5 mM EDTA, 1 mM TCEPplus Complete—EDTA Free Protease inhibitor tablets, 1 tablet/25 ml ofresuspended cells, pH 8.0). The suspension is homogenized and sonicated3×30 sec. NP40, then added to a final concentration of 0.5 % and thetubes are rocked for 30 min at 4° C. Following centrifugation at 11000rpm for 25 to 30 min, the supernatant is incubated with GlutathioneSepharose 4B (Amersham, cat# 17-0756-01) at a ratio of 1 ml of beads per100 ml of original culture volume for 1 to 2 hours at 4° C. with gentlerocking. The beads are pelleted and washed once with 10 bed volumes ofthe lysis buffer, then twice with 10 bed volumes of Prescission Proteasebuffer (50 mM Tris-HCL, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1% NP-40, pH7.0.). Prescission Protease (Amersham, product# 27-0843) is added at aratio of 80 ul (160 Units) per ml of GST resin, and allowed to incubatefor 4 h at 4° C. The supernatant containing the cleaved E2 protein iscollected, and the resin is washed twice with one bed volume ofPrescission buffer. All three fractions are analyzed by SDS-PAGE andpooled when appropriate.

Ubiquitin Production

Ubiquitin was cloned into the pFLAG (DYKDDDDK; SEQ ID NO:13)-MacExpression Vector (Sigma) as a HindIII-EcoRI fragment by PCR. Thisresults in expression of amino-terminal FLAG (DYKDDDDK; SEQ ID NO:13)fusion ubiquitin in E.Coli.

Materials and Methods

The induction of protein expression and cell lysis is similar to theabove GST-E2 preparation, except that the supernatant is loaded over aFLAG (DYKDDDDK; SEQ ID NO:13)-affinity resin (VVR, cat# IB 13020) at aratio of 15 ml of beads per 1 L of original culture. The resin is thenwashed with 10 bed volumes of lysis buffer. The protein is eluted fromthe column with: 100 mM Acetic acid, 10% glycerol, 200 mM NaCl, 2.5 mMEDTA, 0.1% NP-40, pH 3.5. The elutions are collected as 1 bed volumefractions into tubes that contain {fraction (1/10)}^(th) volume of 2 MTris, 80 mM B-ME, pH 9.0 to neutralize the pH. The elution fractions areanalyzed by SDS-PAGE and the appropriate fractions are pooled anddialyzed against 400 volumes of 20 mM Tris, 10% glycerol, 200 mM NaCl,2.5 mMEDTA, pH 8.0.

Production of E3

Coding sequences for E3 complex were also amplified by PCR andbaculoviruses were generated using the Bac-to-Bac system (GibcoBRL). E3contains two subunits, which are expressed by co-infection of the twobaculovirus in the same Hi-5 insect cells. One of the subunit isHis-tagged, with the other associating subunit untagged. The detailprocedure was done following the Bac to Bac Baculovirus Expressionsystem by GibcoBRL. For example, ROC1 was cloned into the pFastBacHtbvector with a N-terminal His6-tag, while CUL1 was insert into thepFastBac1 vector without any fusing tag. After transposition and BacmidDNA transfection into SF-9 cells, Baculoviruses were harvested,amplified, and used to co-infect Hi-5 cells for protein expression.

Materials and Methods

Cells are harvested, washed once with cold PBS, and resuspended in about6 volumes of lysis buffer (20 mM Tris, 20% glycerol, 0.5 M Nacl, 15 mMimidazole, 1 MM TCEP plus Complete—EDTA Free Protease inhibitor tablets,I tablet/25ml of resuspended cells, pH 8.0. ). The suspension is thensonicated 3×30 sec, followed by addition of NP40 to a finalconcentration of 0.5 % and incubation for 30 min at 4° C. The lysate isthen centrifuged and the supernatant is incubated with pre-equilibrated(lysis buffer+NP40) Ni- NTA Agarose beads (Qiagen, cat# 1000632) for 1to 2 hrs. The pelleted beads are washed 2 times with lysis buffer,resuspended in 1 to 2 volumes of lysis buffer and transferred to adisposable column for elution. Elution is accomplished using 5×1-bedvolume aliquots of Lysis buffer+250 mM imidazole. Elution fractions areanalyzed by SDS-PAGE and appropriate fractions are pooled. The elutionpool is then desalted using either a desalting column or a centrifugalconcentration device (more often used for large volumes.) When usingcentrifugal devices, the eluted pool is diluted 1:1 with lysis bufferthat has no imidazole and spun at the appropriate speed until the volumeis reduced by half. At this point an equal volume of fresh buffer isadded and the device is respun. This is done a total of four timesresulting in a 32 fold exchange.

Example 2 Ubiquitin Conjugation Assay

Ubiquitin conjugating activity of E1+E2 was measured using the followingprotocol with FLAG (DYKDDDDK; SEQ ID NO:13)-ubiquin, purified from E.coli, and the E2 Ubch5c, purified as His-Ubch5c from E. coli.

Materials and Methods

The following procedures were used for assays measuring ubiquitinconjugation. The wells of Nickel-substrate 96-well plates (PierceChemical) are blocked with 100 μl of 1% casein/phosphate buffered saline(PBS) for 1 hour at room temperature, then washed with 200 μl of PBST(0.1% Tween-20 in PBS) 3 times. To each well is added the following FLAG(DYKDDDDK; SEQ ID NO:13)-ubiquitin (see above) reaction solution:

Final Concentration

62.5 mM Tris pH 7.5

6.25 mM MgCl₂

0.75 mM DTT

2.5 mM ATP

2.5 mM NaF

12.5 nM Okadaic acid

100 ng FLAG (DYKDDDDK; SEQ ID NO:13)-ubiquitin (made as describedabove).

To the above solution is then added 10 μl of E1,His-E2 in 20 mM Trisbuffer, pH 7.5, and 5% glycerol. His-E2 is made as described above. E1is obtained commercially (Affiniti Research Products, Exeter, U.K.). Thefollowing amounts of each enzyme are used for these assays: 5 ng/well ofE1; 25 nl/well E2. The reaction is then allowed to proceed at roomtemperature for 1 hour.

Following the ubiquitin conjugation reaction, the wells are washed with200 μl of PBST 3 times. For measurement of the enzyme-bound ubiquitin,100 μl of Mouse anti-FLAG (DYKDDDDK; SEQ ID NO:13) (1:10,000) andanti-Mouse Ig-HRP (1:15,000) in PBST are added to each well and allowedto incubate at room temperature for 1 hour. The wells are then washedwith 200 μl of PBST 3 times, followed by the addition of 100 μl ofluminol substrate (1/5 dilution). Luminescence for each well is thenmeasured using a fluorimeter.

Results Ubiquitin Activating and Conjugating Activity

FIG. 1A shows the luminescence measured for E1 alone and for E1+his-E2,as described above.

Example 3 Ubiquitin Ligase Assay

Ubiquitin ligase activity of E1+E2+E3 was measured using the followingprotocol with FLAG (DYKDDDDK; SEQ ID NO:13)-ubiquin, purified from E.coli, the E2 Ubch5c, purified as GST-Ubch5c from E. Coli with the GSTtag removed, and the E3 His-ROC/Cul1 complex purified from Hi-5 cells byBaculovirus co-infection. This assay was also used to show the effectsof candidate modulators on ubiquitin ligase activity.

Materials and Methods

The wells of Nickel-substrate 96-well plates (Pierce Chemical) areblocked with 100 μl of 1 casein/phosphate buffered saline (PBS) for 1hour at room temperature, then washed with 200 μl of PBST (0.1% Tween-20in PBS) 3 times. To each well is added the following FLAG (DYKDDDDK; SEQID NO:13)-ubiquitin (see above) reaction solution

Final Concentration

62.5 mM Tris pH 7.5

6.25 mM MgCl₂

0.75 mM DYT

2.5 mM ATP

2.5 mM NaF

12.5 nM Okadaic acid

100 ng FLAG (DYKDDDDK; SEQ ID NO:13)-ubiquitin (made as describedabove).

The buffer solution is brought to a final volume of 80 pI withmilipore-filtered water. For assays directed to identifying modulatorsof ubiquitin ligase activity, 10 μl of a candidate modulator compound inDMSO is then added to the solution. If no candidate modulator is added,10 μl of DMSO is added to the solution.

To the above solution is then added 10 μl of ubiquitination enzymes in20 mM Tris buffer, pH 7.5, and 5% glycerol. E2-Ubch5c andE3-HisROC1/Cul1 are made as described above. E1 is obtained commercially(Affiniti Research Products, Exeter, U.K.). The following amounts ofeach enzyme are used for these assays: 5 ng/well of E1; 25 nl/well E2;and 100 ng/well His-E3. The reaction is then allowed to proceed at roomtemperature for 1 hour.

Following the ubiquitination reaction, the wells are washed with 200 μlof PBST 3 times. For measurement of the enzyme-bound ubiquitin, 100 μlof Mouse anti-FLAG (DYKDDDDK; SEQ ID NO:13) (1:10,000) and anti-MouseIg-HRP (1:15,000) in PBST are added to each well and allowed to incubateat room temperature for 1 hour. The wells are then washed with 200 μl ofPBST 3 times, followed by the addition of 100 μl of luminol substrate(1/5 dilution). Luminescence for each well is then measured using afluorimeter.

Results Ubiquitin Ligase Activity

FIG. 1 shows the luminescence measured for several differentcombinations of ubiquitination enzymes. In these experiments, only E3was in the form His-E3. The luminescence measurements show that theassay specifically measures the activity of the entire ubiquitinationenzyme cascade, which requires the presence of all three ubiquitinationenzymes in the reaction.

Variations of Composition Components

FIG. 2A shows the relative effect of varying the amount of E1 onubiquitin ligase activity in the above procedure, in presence andabsence of DMSO. The addition of about 10 ng per 100 μl reactionsolution provides maximum ubiquitin ligase activity with the othercomponents of the composition kept as detailed above. The presence ofDMSO does not significantly affect the activity of the ubiquitinationenzymes.

The relative effect of varying E3 and ubiquitin concentration of thereaction composition is shown in FIG. 2B. Generally speaking, maximumubiquitin ligase activity was obtained with 200 to 300 ng per 100 μl ofE3 at each concentration of ubiquitin, while increasing ubiquitinconcentration generally increased ubiquitin ligase activity at eachconcentration of E3.

It was also found that blocking of the wells with 1% casein improved thesignal to noise ratio over either no blocking or blocking with 5% bovineserum albumen (BSA). Background was determined after combining all ofthe components as above except His-E3 and measuring the resultingfluorescence after pre-treating the wells with 5% BSA, 1% casein ornothing. Results are shown in FIG. 3.

Identification of modulators of Ubiquitin Ligase Activity

To show that the assay is useful for identifying modulators of ubiquitinligase activity, several candidate modulators were combined at varyingconcentrations with the assay components as described above. FIG. 4shows the results from two identified modulators of ubiquitin ligaseactivity. The modulators decreased ubiquitin ligase activity in adose-dependent fashion for ubiquitination enzyme compositions comprisingeither ROC1/Cul1 or ROC2/Cul5 as the E3 component.

Comparison of the effect of ubiquitin ligase activity modulators onreaction compositions, as described above, either containing E1, E2 andHis-E3 or containing E1, His-E2 and lacking E3 shows whether themodulator affects E3 or an enzyme other than E3. In FIG. 5A, theidentified modulator decreases ubiquitin ligase activity in the presenceof E3, but does not alter activity in the absence of E3, showing thatthe modulator has a specific effect on E3 ligase activity. In contrast,results shown in FIG. 5B for another modulator reveals that thiscompound reduces activity whether or not E3 is present, showing thatthis modulator affects a member of the ubiquitination enzyme cascadeother than E3.

13 1 3177 DNA Oryctolagus cuniculus 1 atgtccagct cgccgctgtc caagaaacgtcgcgtgtccg ggcctgatcc aaagccgggt 60 tctaactgct cccctgccca gtccgtgttgccccaagtgc cctcggcgcc aaccaacgga 120 atggcgaaga acggcagtga agcagacatcgatgagggcc tttactcccg gcagctgtat 180 gtgttgggcc atgaggcgat gaagcggctccagacatcca gcgttctggt gtcaggcctg 240 cggggcctgg gggtagagat cgcgaagaacatcatccttg gcggggtcaa ggccgtgacc 300 ctccatgacc agggcacggc ccagtgggctgacctctcct cccagttcta cctgcgagag 360 gaggacatag ggaaaaaccg cgctgaggtgtcacagcccc gccttgctga actcaatagc 420 tacgtgcctg tcaccgccta cactgggccgctggttgagg acttcctcag tggcttccag 480 gtggtggtcc tcactaacag ccccctggaggaccagctgc gcgtgggcga gttctgtcat 540 agccgtggca tcaagctggt agtggcagacacgagaggct tgtttgggca actcttctgc 600 gactttggag aggaaatgat cctcacagattccaacgggg agcagcccct cagcaccatg 660 gtttctatgg tcaccaagga caaccctggtgtggttacct gcctggatga ggcccgacat 720 gggtttgaga gtggcgattt tgtttccttctccgaagtac agggcatgac tgagctcaat 780 ggaaaccagc ccatagagat caaagtcctgggtccttaca cctttagcat ctgtgacacc 840 tccaacttct ccgattacat ccgtggaggcattgtcagcc aggtcaaagt acctaagaag 900 ataagcttta aatccttgtc agcctcgctggcagagcctg actttgtgat gacggacttc 960 gccaagtttt ctcgccccgc tcagcttcacattggcttcc aggccttgca caagttctgt 1020 gcacagcaca gccggccacc tagaccccggaacgaggagg atgcagcaga gctggtgacc 1080 ctagcacgcg ctgtgaactc taaagcctcgtcggcagtgc agcaagatag cctggatgag 1140 gacctcatcc ggaacctggc ctttgtggcagccggggacc tggcgcccat caatgccttc 1200 attgggggcc tggctgccca ggaagtcatgaaggcctgct ctgggaagtt tatgcccatc 1260 atgcagtggc tgtactttga tgcccttgagtgtctcccgg aggacaaaga atccctcaca 1320 gaggacaagt gcctcccgcg ccagaaccgttatgatgggc aggtggctgt gtttggctca 1380 gacctgcaag agaagctggg caggcagaagtacttcctgg tgggtgcagg ggctattggc 1440 tgtgagctgc tcaagaactt tgccatgattgggctgggct gtggtgagaa cggagaaata 1500 attgtcacag acatggacac cattgagaaatctaatctga accgacagtt tctattccgg 1560 ccctgggatg tcacgaagtt aaaatctgacacagctgctg cagctgtgca ccagatgaat 1620 ccacatatcc gggtgacaag ccaccagaaccgtgtgggtc ctgacactga acgtatctac 1680 gacgacgatt tcttccaaac tctggatggcgtggccaacg ccttagacaa cgtggatgcc 1740 cgcatgtaca tggaccgccg ctgcgtgtactaccggaagc cgctgctcga atcaggcacc 1800 ctgggcacca agggcaacgt ccaggtggtgatccccttcc tgacagagtc ctacagctcc 1860 agccaagacc cacctgagaa gtccatccccatctgtaccc tgaagaactt ccccaacgcc 1920 atcgaacaca ctcttcagtg ggctcgggatgaatttgaag gcctcttcaa gcagccagcg 1980 gaaaatgtca accagtacct cacagaccctaagtttgtgg agcggacatt gcggctggcg 2040 ggtacccagc cactggaggt gctggaggctgtgcagcgca gcctggtgct gcagctaccg 2100 cagagctggg cagactgtgt gacctgggcctgccaccact ggcacaccca gtattctaac 2160 aatatccggc agctgttgca caacttccctcccgaccagc tcacaagctc gggagctccc 2220 ttctggtctg ggcccaaacg ttgtcctcacccactcacct ttgatgttag caaccctctg 2280 catctggact atgtgatggc tgctgccaacctgtttgccc agacctacgg gctggcaggc 2340 tctcaggacc gagctgctgt ggccacactcctgcagtctg tacaggtccc cgagtttacc 2400 cccaagtctg gcgtcaaaat ccacgtttctgaccaggagc tgcagagcgc caatgcttct 2460 gttgacgaca gccgtttaga ggagctcaaggctacgctgc ctagccccga caagctccct 2520 ggattcaaga tgtaccccat tgactttgagaaggatgatg atagtaactt tcacatggac 2580 ttcattgtgg ccgcatccaa cctccgggccgaaaactatg acattccccc tgcagaccgg 2640 cacaagagca agctgattgc agggaagatcatcccagcca ttgccacgac cacagcagct 2700 gtcgttggcc ttgtgtgtct ggagctgtacaaggtagtgc agggacaccg acacctcgac 2760 tcctacaaga atggtttcct caacctggccctgccgtttt tcggtttctc tgaacctctg 2820 gctgcaccac gtcaccagta ctataaccaagagtggacat tgtgggatcg ctttgaggtt 2880 cagggactgc agcccaacgg tgaggagatgaccctcaaac aattcctcga ctactttaag 2940 acagagcaca aattggagat taccatgctgtcccagggtg tgtccatgct ctattccttc 3000 tttatgccag ctgcgaagct caaggaacggttggaccagc cgatgacaga gattgtaagc 3060 cgtgtgtcga agcgaaagct gggccgccacgtgcgggcgc tggtgcttga gctgtgctgc 3120 aacgacgaga gcggcgagga cgtcgaagtcccctacgtcc gatataccat ccgttaa 3177 2 1059 PRT Oryctolagus cuniculus 2Met Ser Ser Ser Pro Leu Ser Lys Lys Arg Arg Val Ser Gly Pro Asp 1 5 1015 Pro Lys Pro Gly Ser Asn Cys Ser Pro Ala Gln Ser Val Leu Pro Gln 20 2530 Val Pro Ser Ala Pro Thr Asn Gly Met Ala Lys Asn Gly Ser Glu Ala 35 4045 Asp Ile Asp Glu Gly Leu Tyr Ser Arg Gln Leu Tyr Val Leu Gly His 50 5560 Glu Ala Met Lys Arg Leu Gln Thr Ser Ser Val Leu Val Ser Gly Leu 65 7075 80 Arg Gly Leu Gly Val Glu Ile Ala Lys Asn Ile Ile Leu Gly Gly Val 8590 95 Lys Ala Val Thr Leu His Asp Gln Gly Thr Ala Gln Trp Ala Asp Leu100 105 110 Ser Ser Gln Phe Tyr Leu Arg Glu Glu Asp Ile Gly Lys Asn ArgAla 115 120 125 Glu Val Ser Gln Pro Arg Leu Ala Glu Leu Asn Ser Tyr ValPro Val 130 135 140 Thr Ala Tyr Thr Gly Pro Leu Val Glu Asp Phe Leu SerGly Phe Gln 145 150 155 160 Val Val Val Leu Thr Asn Ser Pro Leu Glu AspGln Leu Arg Val Gly 165 170 175 Glu Phe Cys His Ser Arg Gly Ile Lys LeuVal Val Ala Asp Thr Arg 180 185 190 Gly Leu Phe Gly Gln Leu Phe Cys AspPhe Gly Glu Glu Met Ile Leu 195 200 205 Thr Asp Ser Asn Gly Glu Gln ProLeu Ser Thr Met Val Ser Met Val 210 215 220 Thr Lys Asp Asn Pro Gly ValVal Thr Cys Leu Asp Glu Ala Arg His 225 230 235 240 Gly Phe Glu Ser GlyAsp Phe Val Ser Phe Ser Glu Val Gln Gly Met 245 250 255 Thr Glu Leu AsnGly Asn Gln Pro Ile Glu Ile Lys Val Leu Gly Pro 260 265 270 Tyr Thr PheSer Ile Cys Asp Thr Ser Asn Phe Ser Asp Tyr Ile Arg 275 280 285 Gly GlyIle Val Ser Gln Val Lys Val Pro Lys Lys Ile Ser Phe Lys 290 295 300 SerLeu Ser Ala Ser Leu Ala Glu Pro Asp Phe Val Met Thr Asp Phe 305 310 315320 Ala Lys Phe Ser Arg Pro Ala Gln Leu His Ile Gly Phe Gln Ala Leu 325330 335 His Lys Phe Cys Ala Gln His Ser Arg Pro Pro Arg Pro Arg Asn Glu340 345 350 Glu Asp Ala Ala Glu Leu Val Thr Leu Ala Arg Ala Val Asn SerLys 355 360 365 Ala Ser Ser Ala Val Gln Gln Asp Ser Leu Asp Glu Asp LeuIle Arg 370 375 380 Asn Leu Ala Phe Val Ala Ala Gly Asp Leu Ala Pro IleAsn Ala Phe 385 390 395 400 Ile Gly Gly Leu Ala Ala Gln Glu Val Met LysAla Cys Ser Gly Lys 405 410 415 Phe Met Pro Ile Met Gln Trp Leu Tyr PheAsp Ala Leu Glu Cys Leu 420 425 430 Pro Glu Asp Lys Glu Ser Leu Thr GluAsp Lys Cys Leu Pro Arg Gln 435 440 445 Asn Arg Tyr Asp Gly Gln Val AlaVal Phe Gly Ser Asp Leu Gln Glu 450 455 460 Lys Leu Gly Arg Gln Lys TyrPhe Leu Val Gly Ala Gly Ala Ile Gly 465 470 475 480 Cys Glu Leu Leu LysAsn Phe Ala Met Ile Gly Leu Gly Cys Gly Glu 485 490 495 Asn Gly Glu IleIle Val Thr Asp Met Asp Thr Ile Glu Lys Ser Asn 500 505 510 Leu Asn ArgGln Phe Leu Phe Arg Pro Trp Asp Val Thr Lys Leu Lys 515 520 525 Ser AspThr Ala Ala Ala Ala Val His Gln Met Asn Pro His Ile Arg 530 535 540 ValThr Ser His Gln Asn Arg Val Gly Pro Asp Thr Glu Arg Ile Tyr 545 550 555560 Asp Asp Asp Phe Phe Gln Thr Leu Asp Gly Val Ala Asn Ala Leu Asp 565570 575 Asn Val Asp Ala Arg Met Tyr Met Asp Arg Arg Cys Val Tyr Tyr Arg580 585 590 Lys Pro Leu Leu Glu Ser Gly Thr Leu Gly Thr Lys Gly Asn ValGln 595 600 605 Val Val Ile Pro Phe Leu Thr Glu Ser Tyr Ser Ser Ser GlnAsp Pro 610 615 620 Pro Glu Lys Ser Ile Pro Ile Cys Thr Leu Lys Asn PhePro Asn Ala 625 630 635 640 Ile Glu His Thr Leu Gln Trp Ala Arg Asp GluPhe Glu Gly Leu Phe 645 650 655 Lys Gln Pro Ala Glu Asn Val Asn Gln TyrLeu Thr Asp Pro Lys Phe 660 665 670 Val Glu Arg Thr Leu Arg Leu Ala GlyThr Gln Pro Leu Glu Val Leu 675 680 685 Glu Ala Val Gln Arg Ser Leu ValLeu Gln Leu Pro Gln Ser Trp Ala 690 695 700 Asp Cys Val Thr Trp Ala CysHis His Trp His Thr Gln Tyr Ser Asn 705 710 715 720 Asn Ile Arg Gln LeuLeu His Asn Phe Pro Pro Asp Gln Leu Thr Ser 725 730 735 Ser Gly Ala ProPhe Trp Ser Gly Pro Lys Arg Cys Pro His Pro Leu 740 745 750 Thr Phe AspVal Ser Asn Pro Leu His Leu Asp Tyr Val Met Ala Ala 755 760 765 Ala AsnLeu Phe Ala Gln Thr Tyr Gly Leu Ala Gly Ser Gln Asp Arg 770 775 780 AlaAla Val Ala Thr Leu Leu Gln Ser Val Gln Val Pro Glu Phe Thr 785 790 795800 Pro Lys Ser Gly Val Lys Ile His Val Ser Asp Gln Glu Leu Gln Ser 805810 815 Ala Asn Ala Ser Val Asp Asp Ser Arg Leu Glu Glu Leu Lys Ala Thr820 825 830 Leu Pro Ser Pro Asp Lys Leu Pro Gly Phe Lys Met Tyr Pro IleAsp 835 840 845 Phe Glu Lys Asp Asp Asp Ser Asn Phe His Met Asp Phe IleVal Ala 850 855 860 Ala Ser Asn Leu Arg Ala Glu Asn Tyr Asp Ile Pro ProAla Asp Arg 865 870 875 880 His Lys Ser Lys Leu Ile Ala Gly Lys Ile IlePro Ala Ile Ala Thr 885 890 895 Thr Thr Ala Ala Val Val Gly Leu Val CysLeu Glu Leu Tyr Lys Val 900 905 910 Val Gln Gly His Arg His Leu Asp SerTyr Lys Asn Gly Phe Leu Asn 915 920 925 Leu Ala Leu Pro Phe Phe Gly PheSer Glu Pro Leu Ala Ala Pro Arg 930 935 940 His Gln Tyr Tyr Asn Gln GluTrp Thr Leu Trp Asp Arg Phe Glu Val 945 950 955 960 Gln Gly Leu Gln ProAsn Gly Glu Glu Met Thr Leu Lys Gln Phe Leu 965 970 975 Asp Tyr Phe LysThr Glu His Lys Leu Glu Ile Thr Met Leu Ser Gln 980 985 990 Gly Val SerMet Leu Tyr Ser Phe Phe Met Pro Ala Ala Lys Leu Lys 995 1000 1005 GluArg Leu Asp Gln Pro Met Thr Glu Ile Val Ser Arg Val Ser 1010 1015 1020Lys Arg Lys Leu Gly Arg His Val Arg Ala Leu Val Leu Glu Leu 1025 10301035 Cys Cys Asn Asp Glu Ser Gly Glu Asp Val Glu Val Pro Tyr Val 10401045 1050 Arg Tyr Thr Ile Arg Glx 1055 3 444 DNA Homo sapiens 3atggcgctga aacggattaa taaggaactt agtgatttgg cccgtgaccc tccagcacaa 60tgttctgcag gtccagttgg ggatgatatg tttcattggc aagccacaat tatgggacct 120aatgacagcc catatcaagg cggtgtattc tttttgacaa ttcattttcc tacagactac 180cccttcaaac cacctaaggt tgcatttaca acaagaattt atcatccaaa tattaacagt 240aatggcagca tttgtctcga tattctaaga tcacagtggt cgcctgcttt aacaatttct 300aaagttcttt tatccatttg ttcactgcta tgtgatccaa acccagatga ccccctagtg 360ccagagattg cacggatcta taaaacagac agagataagt acaacagaat atctcgggaa 420tggactcaga agtatgccat gtga 444 4 148 PRT Homo sapiens 4 Met Ala Leu LysArg Ile Asn Lys Glu Leu Ser Asp Leu Ala Arg Asp 1 5 10 15 Pro Pro AlaGln Cys Ser Ala Gly Pro Val Gly Asp Asp Met Phe His 20 25 30 Trp Gln AlaThr Ile Met Gly Pro Asn Asp Ser Pro Tyr Gln Gly Gly 35 40 45 Val Phe PheLeu Thr Ile His Phe Pro Thr Asp Tyr Pro Phe Lys Pro 50 55 60 Pro Lys ValAla Phe Thr Thr Arg Ile Tyr His Pro Asn Ile Asn Ser 65 70 75 80 Asn GlySer Ile Cys Leu Asp Ile Leu Arg Ser Gln Trp Ser Pro Ala 85 90 95 Leu ThrIle Ser Lys Val Leu Leu Ser Ile Cys Ser Leu Leu Cys Asp 100 105 110 ProAsn Pro Asp Asp Pro Leu Val Pro Glu Ile Ala Arg Ile Tyr Lys 115 120 125Thr Asp Arg Asp Lys Tyr Asn Arg Ile Ser Arg Glu Trp Thr Gln Lys 130 135140 Tyr Ala Met Glx 145 5 84 PRT Homo sapiens 5 Met Lys Val Lys Ile LysCys Trp Asn Gly Val Ala Thr Trp Leu Trp 1 5 10 15 Val Ala Asn Asp GluAsn Cys Gly Ile Cys Arg Met Ala Phe Asn Gly 20 25 30 Cys Cys Pro Asp CysLys Val Pro Gly Asp Asp Cys Pro Leu Val Trp 35 40 45 Gly Gln Cys Ser HisCys Phe His Met His Cys Ile Leu Lys Trp Leu 50 55 60 His Ala Gln Gln ValGln Gln His Cys Pro Met Cys Arg Gln Thr Trp 65 70 75 80 Lys Phe Lys Glu6 108 PRT Homo sapiens 6 Met Ala Ala Ala Met Asp Val Asp Thr Pro Ser GlyThr Asn Ser Gly 1 5 10 15 Ala Gly Lys Lys Arg Phe Glu Val Lys Lys TrpAsn Ala Val Ala Leu 20 25 30 Trp Ala Trp Asp Ile Val Val Asp Asn Cys AlaIle Cys Arg Asn His 35 40 45 Ile Met Asp Leu Cys Ile Glu Cys Gln Ala AsnGln Ala Ser Ala Thr 50 55 60 Ser Glu Glu Cys Thr Val Ala Trp Gly Val CysAsn His Ala Phe His 65 70 75 80 Phe His Cys Ile Ser Arg Trp Leu Lys ThrArg Gln Val Cys Pro Leu 85 90 95 Asp Asn Arg Glu Trp Glu Phe Gln Lys TyrGly His 100 105 7 342 DNA Homo sapiens 7 atggccgacg tggaagacggagaggaaacc tgcgccctgg cctctcactc cgggagctca 60 ggctcaacgt cgggaggcgacaagatgttc tccctcaaga agtggaaccc ggtggccatg 120 tggagctggg acgtggagtgcgatacgtgc gccatctgca gggtccaggt gatggatgcc 180 tgtcttagat gtcaagctgaaaacaaacaa gaggactgtg ttgtggtctg gggagaatgt 240 aatcattcct tccacaactgctgcatgtcc ctgtgggtga aacagaacaa tcgctgccct 300 ctctgccagc aggactgggtggtccaaaga atcggcaaat ga 342 8 113 PRT Homo sapiens 8 Met Ala Asp ValGlu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His 1 5 10 15 Ser Gly SerSer Gly Ser Thr Ser Gly Gly Asp Lys Met Phe Ser Leu 20 25 30 Lys Lys TrpAsn Pro Val Ala Met Trp Ser Trp Asp Val Glu Cys Asp 35 40 45 Thr Cys AlaIle Cys Arg Val Gln Val Met Asp Ala Cys Leu Arg Cys 50 55 60 Gln Ala GluAsn Lys Gln Glu Asp Cys Val Val Val Trp Gly Glu Cys 65 70 75 80 Asn HisSer Phe His Asn Cys Cys Met Ser Leu Trp Val Lys Gln Asn 85 90 95 Asn ArgCys Pro Leu Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly 100 105 110 Lys9 2343 DNA Homo sapiens 9 atggcgacgt ctaatctgtt aaagaataaa ggttctcttcagtttgaaga caaatgggat 60 tttatgcgcc cgattgtttt gaagctttta cgccaggaatctgttacaaa acagcagtgg 120 tttgatctgt tttcggatgt gcatgcagtc tgtctttgggatgataaagg cccagcaaaa 180 attcatcagg ctttaaaaga agatattctt gagtttattaagcaggcaca ggcacgagta 240 ctgagccatc aagatgatac ggctttgcta aaagcatatattgttgaatg gcgaaagttc 300 tttacacaat gtgatatttt accaaaacct ttttgtcaactagagattac tttaatgggt 360 aaacagggca gcaataaaaa atcaaatgtg gaagacagtattgttcgaaa gcttatgctt 420 gatacatgga atgagtcaat cttttcaaac ataaaaaacagactccaaga tagtgcaatg 480 aagctggtac atgctgagag attgggagaa gcttttgattctcagctggt tattggagta 540 agagaatcct atgttaacct ttgttctaat cctgaggataaacttcaaat ttatagggac 600 aattttgaga aggcatactt ggattcaaca gagagattttatagaacaca agcaccctcg 660 tatttacaac caaatggtgt acagaattat atgaaatatgcagatgctaa attaaaagaa 720 gaagaaaaac gagcactacg ttatttagaa acaagacgagaatgtaactc cgttgaagca 780 ctcatggaat gctgtgtaaa tgccctggtg acatcatttaaagagactat cttagctgag 840 tgccaaggca tgatcaagag aaatgaaact gaaaaattacatttaatgtt ttcattgatg 900 gacaaagttc ctaatggtat agagccaatg ttgaaagacttggaggaaca tatcattagt 960 gctggcctgg cagatatggt agcagctgct gaaactattactactgactc tgagaaatac 1020 gttgagcagt tacttacact atttaataga tttagtaaactcgtcaaaga agcttttcaa 1080 gatgatccac gatttcttac tgcaagagat aaggcgtataaagcagttgt taatgatgct 1140 accatattta aacttgaatt acctttgaag cagaagggggtgggattaaa aactcagcct 1200 gaatcaaaat gccctgagct gcttgccaat tactgtgacatgttgctaag aaaaacacca 1260 ttaagcaaaa aactaacctc tgaagagatt gaagcaaagcttaaagaagt gctcttggta 1320 cttaagtatg tacagaacaa agatgttttt atgaggtatcataaagctca tttgacacga 1380 cgtcttatat tagacatctc tgccgatagt gaaattgaagaaaacatggt agagtggcta 1440 agagaagttg gtatgccagc ggattatgta aacaagcttgctagaatgtt tcaggacata 1500 aaagtatctg aagatttgaa ccaagctttt aaggaaatgcacaaaaataa taaattggca 1560 ttaccagctg attcagttaa tataaaaatt ctgaatgctggcgcctggtc aagaagttct 1620 gagaaagtct ttgtctcact tcctactgaa ctggaggacttgataccgga agtagaagaa 1680 ttctacaaaa aaaatcatag tggtagaaaa ttacattggcatcatctcat gtcaaatgga 1740 attataacat ttaagaatga agttggtcaa tatgatttggaggtaaccac gtttcagctc 1800 gctgtattgt ttgcatggaa ccaaagaccc agagagaaaatcagctttga aaatcttaag 1860 cttgcaactg aactccctga tgctgaactt aggaggactttatggtcttt agtagctttc 1920 ccaaaactca aacggcaagt ttttttgtat gaccctcaagtcaactcacc caaagacttt 1980 acagaaggta ccctcttctc agtgaaccag gagttcagtttaataaaaaa tgcaaaggtt 2040 cagaaaaggg gtaaaatcaa cttgattgga cgtttgcagctcactacaga aaggatgaga 2100 gaagaagaga atgaaggaat agttcaacta cgaatactaagaacccagga agctatcata 2160 caaataatga aaatgagaaa gaaaattagt aatgctcagctgcagactga attagtagaa 2220 attttgaaaa acatgttctt gccacaaaag aaaatgataaaagagcaaat agagtggcta 2280 atagagcaca aatacatcag aagagatgaa tctgatatcaacactttcat atatatggca 2340 taa 2343 10 861 PRT Homo sapiens 10 Met ArgSer Phe Ala Trp Gly Ser Ser Gly Asp His Val Gly Asp Lys 1 5 10 15 SerGlu Glu Ala Pro Gly Ala Trp Asp Glu Val Ser Ala Val Gly Ala 20 25 30 LeuLeu Gln Arg Pro Pro His Pro Gly Ala Gly Pro Thr Gly Pro Gly 35 40 45 ProTrp Trp Glu Leu Arg Pro Pro Val Lys Ala Trp Pro Gly Arg Glu 50 55 60 ArgHis Glu Phe Ser Arg Arg Leu Val Ser Arg Glu Ser Lys Leu Lys 65 70 75 80Asn Met Ala Thr Ser Asn Leu Leu Lys Asn Lys Gly Ser Leu Gln Phe 85 90 95Glu Asp Lys Trp Asp Phe Met Arg Pro Ile Val Leu Lys Leu Leu Arg 100 105110 Gln Glu Ser Val Thr Lys Gln Gln Trp Phe Asp Leu Phe Ser Asp Val 115120 125 His Ala Val Cys Leu Trp Asp Asp Lys Gly Pro Ala Lys Ile His Gln130 135 140 Ala Leu Lys Glu Asp Ile Leu Glu Phe Ile Lys Gln Ala Gln AlaArg 145 150 155 160 Val Leu Ser His Gln Asp Asp Thr Ala Leu Leu Lys AlaTyr Ile Val 165 170 175 Glu Trp Arg Lys Phe Phe Thr Gln Cys Asp Ile LeuPro Lys Pro Phe 180 185 190 Cys Gln Leu Glu Ile Thr Leu Met Gly Lys GlnGly Ser Asn Lys Lys 195 200 205 Ser Asn Val Glu Asp Ser Ile Val Arg LysLeu Met Leu Asp Thr Trp 210 215 220 Asn Glu Ser Ile Phe Ser Asn Ile LysAsn Arg Leu Gln Asp Ser Ala 225 230 235 240 Met Lys Leu Val His Ala GluArg Leu Gly Glu Ala Phe Asp Ser Gln 245 250 255 Leu Val Ile Gly Val ArgGlu Ser Tyr Val Asn Leu Cys Ser Asn Pro 260 265 270 Glu Asp Lys Leu GlnIle Tyr Arg Asp Asn Phe Glu Lys Ala Tyr Leu 275 280 285 Asp Ser Thr GluArg Phe Tyr Arg Thr Gln Ala Pro Ser Tyr Leu Gln 290 295 300 Pro Asn GlyVal Gln Asn Tyr Met Lys Tyr Ala Asp Ala Lys Leu Lys 305 310 315 320 GluGlu Glu Lys Arg Ala Leu Arg Tyr Leu Glu Thr Arg Arg Glu Cys 325 330 335Asn Ser Val Glu Ala Leu Met Glu Cys Cys Val Asn Ala Leu Val Thr 340 345350 Ser Phe Lys Glu Thr Ile Leu Ala Glu Cys Gln Gly Met Ile Lys Arg 355360 365 Asn Glu Thr Glu Lys Leu His Leu Met Phe Ser Leu Met Asp Lys Val370 375 380 Pro Asn Gly Ile Glu Pro Met Leu Lys Asp Leu Glu Glu His IleIle 385 390 395 400 Ser Ala Gly Leu Ala Asp Met Val Ala Ala Ala Glu ThrIle Thr Thr 405 410 415 Asp Ser Glu Lys Tyr Val Glu Gln Leu Leu Thr LeuPhe Asn Arg Phe 420 425 430 Ser Lys Leu Val Lys Glu Ala Phe Gln Asp AspPro Arg Phe Leu Thr 435 440 445 Ala Arg Asp Lys Ala Tyr Lys Ala Val ValAsn Asp Ala Thr Ile Phe 450 455 460 Lys Leu Glu Leu Pro Leu Lys Gln LysGly Val Gly Leu Lys Thr Gln 465 470 475 480 Pro Glu Ser Lys Cys Pro GluLeu Leu Ala Asn Tyr Cys Asp Met Leu 485 490 495 Leu Arg Lys Thr Pro LeuSer Lys Lys Leu Thr Ser Glu Glu Ile Glu 500 505 510 Ala Lys Leu Lys GluVal Leu Leu Val Leu Lys Tyr Val Gln Asn Lys 515 520 525 Asp Val Phe MetArg Tyr His Lys Ala His Leu Thr Arg Arg Leu Ile 530 535 540 Leu Asp IleSer Ala Asp Ser Glu Ile Glu Glu Asn Met Val Glu Trp 545 550 555 560 LeuArg Glu Val Gly Met Pro Ala Asp Tyr Val Asn Lys Leu Ala Arg 565 570 575Met Phe Gln Asp Ile Lys Val Ser Glu Asp Leu Asn Gln Ala Phe Lys 580 585590 Glu Met His Lys Asn Asn Lys Leu Ala Leu Pro Ala Asp Ser Val Asn 595600 605 Ile Lys Ile Leu Asn Ala Gly Ala Trp Ser Arg Ser Ser Glu Lys Val610 615 620 Phe Val Ser Leu Pro Thr Glu Leu Glu Asp Leu Ile Pro Glu ValGlu 625 630 635 640 Glu Phe Tyr Lys Lys Asn His Ser Gly Arg Lys Leu HisTrp His His 645 650 655 Leu Met Ser Asn Gly Ile Ile Thr Phe Lys Asn GluVal Gly Gln Tyr 660 665 670 Asp Leu Glu Val Thr Thr Phe Gln Leu Ala ValLeu Phe Ala Trp Asn 675 680 685 Gln Arg Pro Arg Glu Lys Ile Ser Phe GluAsn Leu Lys Leu Ala Thr 690 695 700 Glu Leu Pro Asp Ala Glu Leu Arg ArgThr Leu Trp Ser Leu Val Ala 705 710 715 720 Phe Pro Lys Leu Lys Arg GlnVal Phe Leu Tyr Asp Pro Gln Val Asn 725 730 735 Ser Pro Lys Asp Phe ThrGlu Gly Thr Leu Phe Ser Val Asn Gln Glu 740 745 750 Phe Ser Leu Ile LysAsn Ala Lys Val Gln Lys Arg Gly Lys Ile Asn 755 760 765 Leu Ile Gly ArgLeu Gln Leu Thr Thr Glu Arg Met Arg Glu Glu Glu 770 775 780 Asn Glu GlyIle Val Gln Leu Arg Ile Leu Arg Thr Gln Glu Ala Ile 785 790 795 800 IleGln Ile Met Lys Met Arg Lys Lys Ile Ser Asn Ala Gln Leu Gln 805 810 815Thr Glu Leu Val Glu Ile Leu Lys Asn Met Phe Leu Pro Gln Lys Lys 820 825830 Met Ile Lys Glu Gln Ile Glu Trp Leu Ile Glu His Lys Tyr Ile Arg 835840 845 Arg Asp Glu Ser Asp Ile Asn Thr Phe Ile Tyr Met Ala 850 855 86011 2469 DNA Homo sapiens 11 atggcggcgg cagttgtggt ggcggagggg gacagcgactcccggcccgg acaggagttg 60 ttagtggcct ggaacaccgt gagcaccggc ctggtgccgccggctgcgct ggggctggtg 120 tcttcccgga ccagcggtgc agtcccgcca aaggaagaggagctccgggc ggcggtggag 180 gttctgaggg gccacgggct acactcggtc ctggaggagtggttcgtgga ggtgctgcag 240 aacgatctgc aggccaacat ctcccctgag ttctggaatgccatctccca atgcgagaac 300 tctgcggatg agccccagtg ccttttgcta ctccttgacgcttttggcct gctggagagc 360 cgcctggatc cctacctgcg tagcctagag ctgctggagaaatggactcg cctgggcttg 420 ctgatgggca ctggtgctca ggggctgcga gaagaagtccacactatgtt gcgcggagtc 480 ttgttcttta gcacccccag aaccttccaa gagatgatccagcgtctgta tgggtgcttc 540 ttgagagtct atatgcagag taagaggaag ggggaagggggcacagaccc ggaactggaa 600 ggggagctgg acagccggta tgcccgtcgc cggtactaccggctcctgca gagcccgctg 660 tgtgcagggt gcagcagtga caagcaacag tgctggtgtcgccaggctct ggagcagttc 720 catcagctca gccaggtctt acacaggctc agtctgctggagcgggtcag tgccgaggct 780 gtgaccacca ccctgcacca ggtgacccgg gagaggatggaggaccgttg ccggggcgag 840 tacgagcgct ccttcctgcg tgagttccac aagtggatcgagcgggtggt cggctggctc 900 ggcaaggtgt tcctgcagga cggccccgcc aggcccgcatctcccgaggc cggcaacacc 960 ctgcgccgct ggcgctgcca cgtgcaaagg ttcttctaccgcatctacgc cagcctgcgc 1020 atcgaggagc tcttcagcat cgtccgagac ttcccagactcccggccagc catcgaggac 1080 ctcaagtact gcctggagag gacggaccag aggcagcagctgctcgtgtc cctcaaggct 1140 gccctggaga ctcggctcct gcatccaggc gtcaacacgtgtgacatcat caccctctat 1200 atctctgcca tcaaggcgct gcgcgtgctg gacccttccatggtcatcct ggaggtggcc 1260 tgtgagccta tccgccgcta cctgaggacg cgggaggacacagtgcggca gattgtggct 1320 gggctgacgg gggactcgga cgggacaggg gacctggctgttgagctgtc caagaccgac 1380 ccggcgagcc tggagacagg ccaggacagt gaggatgactcaggcgagcc agaggactgg 1440 gtcccggacc ctgtggatgc cgatccaggg aagtcgagctccaagcggcg ttcatcggac 1500 atcatcagcc tgctggtcag catctacggc agcaaggacctcttcatcaa tgagtaccgc 1560 tcgctgctgg ccgaccgcct gctgcaccag ttcagcttcagccccgagcg ggagatccgc 1620 aacgtggagc tgctgaagct gcgctttggc gaggccccaatgcacttctg tgaagtcatg 1680 ctgaaggaca tggcggactc ccgccgcatc aatgccaacatccgggagga ggatgagaag 1740 cggccagcag aggagcagcc accgttcggg gtctacgctgtcatcctgtc cagtgagttc 1800 tggccgccct tcaaggacga gaagctggag gtccccgaggatatcagggc agccctggag 1860 gcttactgca agaagtatga gcagctcaag gccatgcggaccctcagttg gaagcacacc 1920 ctgggcctgg tgaccatgga cgtggagctg gccgaccgcacgctgtctgt ggcggtcacc 1980 ccagtacagg cggtgatctt gctgtatttt caggaccaagccagctggac cctggaggaa 2040 ctgagcaagg cggtgaaaat gcccgtggcg ctgctgcggcggcggatgtc cgtgtggctg 2100 cagcagggtg tgctgcgtga ggagcccccc ggcaccttctctgtcattga ggaggagcgg 2160 cctcaggacc gggacaacat ggtgctcatt gacagtgacgacgagagcga ctccggcatg 2220 gcctcccagg ccgaccagaa ggaggaggag ctgctgctcttctggacgta catccaggcc 2280 atgctgacca acctggagag cctctcactg gatcgtatctacaacatgct ccgcatgttt 2340 gtggtgactg ggcctgcact ggccgagatt gacctgcaggagctgcaggg ctacctgcag 2400 aagaaggtgc gggaccagca gctcgtctac tcggccggcgtctaccgcct gcccaagaac 2460 tgcagctga 2469 12 822 PRT Homo sapiens 12 MetAla Ala Ala Val Val Val Ala Glu Gly Asp Ser Asp Ser Arg Pro 1 5 10 15Gly Gln Glu Leu Leu Val Ala Trp Asn Thr Val Ser Thr Gly Leu Val 20 25 30Pro Pro Ala Ala Leu Gly Leu Val Ser Ser Arg Thr Ser Gly Ala Val 35 40 45Pro Pro Lys Glu Glu Glu Leu Arg Ala Ala Val Glu Val Leu Arg Gly 50 55 60His Gly Leu His Ser Val Leu Glu Glu Trp Phe Val Glu Val Leu Gln 65 70 7580 Asn Asp Leu Gln Ala Asn Ile Ser Pro Glu Phe Trp Asn Ala Ile Ser 85 9095 Gln Cys Glu Asn Ser Ala Asp Glu Pro Gln Cys Leu Leu Leu Leu Leu 100105 110 Asp Ala Phe Gly Leu Leu Glu Ser Arg Leu Asp Pro Tyr Leu Arg Ser115 120 125 Leu Glu Leu Leu Glu Lys Trp Thr Arg Leu Gly Leu Leu Met GlyThr 130 135 140 Gly Ala Gln Gly Leu Arg Glu Glu Val His Thr Met Leu ArgGly Val 145 150 155 160 Leu Phe Phe Ser Thr Pro Arg Thr Phe Gln Glu MetIle Gln Arg Leu 165 170 175 Tyr Gly Cys Phe Leu Arg Val Tyr Met Gln SerLys Arg Lys Gly Glu 180 185 190 Gly Gly Thr Asp Pro Glu Leu Glu Gly GluLeu Asp Ser Arg Tyr Ala 195 200 205 Arg Arg Arg Tyr Tyr Arg Leu Leu GlnSer Pro Leu Cys Ala Gly Cys 210 215 220 Ser Ser Asp Lys Gln Gln Cys TrpCys Arg Gln Ala Leu Glu Gln Phe 225 230 235 240 His Gln Leu Ser Gln ValLeu His Arg Leu Ser Leu Leu Glu Arg Val 245 250 255 Ser Ala Glu Ala ValThr Thr Thr Leu His Gln Val Thr Arg Glu Arg 260 265 270 Met Glu Asp ArgCys Arg Gly Glu Tyr Glu Arg Ser Phe Leu Arg Glu 275 280 285 Phe His LysTrp Ile Glu Arg Val Val Gly Trp Leu Gly Lys Val Phe 290 295 300 Leu GlnAsp Gly Pro Ala Arg Pro Ala Ser Pro Glu Ala Gly Asn Thr 305 310 315 320Leu Arg Arg Trp Arg Cys His Val Gln Arg Phe Phe Tyr Arg Ile Tyr 325 330335 Ala Ser Leu Arg Ile Glu Glu Leu Phe Ser Ile Val Arg Asp Phe Pro 340345 350 Asp Ser Arg Pro Ala Ile Glu Asp Leu Lys Tyr Cys Leu Glu Arg Thr355 360 365 Asp Gln Arg Gln Gln Leu Leu Val Ser Leu Lys Ala Ala Leu GluThr 370 375 380 Arg Leu Leu His Pro Gly Val Asn Thr Cys Asp Ile Ile ThrLeu Tyr 385 390 395 400 Ile Ser Ala Ile Lys Ala Leu Arg Val Leu Asp ProSer Met Val Ile 405 410 415 Leu Glu Val Ala Cys Glu Pro Ile Arg Arg TyrLeu Arg Thr Arg Glu 420 425 430 Asp Thr Val Arg Gln Ile Val Ala Gly LeuThr Gly Asp Ser Asp Gly 435 440 445 Thr Gly Asp Leu Ala Val Glu Leu SerLys Thr Asp Pro Ala Ser Leu 450 455 460 Glu Thr Gly Gln Asp Ser Glu AspAsp Ser Gly Glu Pro Glu Asp Trp 465 470 475 480 Val Pro Asp Pro Val AspAla Asp Pro Gly Lys Ser Ser Ser Lys Arg 485 490 495 Arg Ser Ser Asp IleIle Ser Leu Leu Val Ser Ile Tyr Gly Ser Lys 500 505 510 Asp Leu Phe IleAsn Glu Tyr Arg Ser Leu Leu Ala Asp Arg Leu Leu 515 520 525 His Gln PheSer Phe Ser Pro Glu Arg Glu Ile Arg Asn Val Glu Leu 530 535 540 Leu LysLeu Arg Phe Gly Glu Ala Pro Met His Phe Cys Glu Val Met 545 550 555 560Leu Lys Asp Met Ala Asp Ser Arg Arg Ile Asn Ala Asn Ile Arg Glu 565 570575 Glu Asp Glu Lys Arg Pro Ala Glu Glu Gln Pro Pro Phe Gly Val Tyr 580585 590 Ala Val Ile Leu Ser Ser Glu Phe Trp Pro Pro Phe Lys Asp Glu Lys595 600 605 Leu Glu Val Pro Glu Asp Ile Arg Ala Ala Leu Glu Ala Tyr CysLys 610 615 620 Lys Tyr Glu Gln Leu Lys Ala Met Arg Thr Leu Ser Trp LysHis Thr 625 630 635 640 Leu Gly Leu Val Thr Met Asp Val Glu Leu Ala AspArg Thr Leu Ser 645 650 655 Val Ala Val Thr Pro Val Gln Ala Val Ile LeuLeu Tyr Phe Gln Asp 660 665 670 Gln Ala Ser Trp Thr Leu Glu Glu Leu SerLys Ala Val Lys Met Pro 675 680 685 Val Ala Leu Leu Arg Arg Arg Met SerVal Trp Leu Gln Gln Gly Val 690 695 700 Leu Arg Glu Glu Pro Pro Gly ThrPhe Ser Val Ile Glu Glu Glu Arg 705 710 715 720 Pro Gln Asp Arg Asp AsnMet Val Leu Ile Asp Ser Asp Asp Glu Ser 725 730 735 Asp Ser Gly Met AlaSer Gln Ala Asp Gln Lys Glu Glu Glu Leu Leu 740 745 750 Leu Phe Trp ThrTyr Ile Gln Ala Met Leu Thr Asn Leu Glu Ser Leu 755 760 765 Ser Leu AspArg Ile Tyr Asn Met Leu Arg Met Phe Val Val Thr Gly 770 775 780 Pro AlaLeu Ala Glu Ile Asp Leu Gln Glu Leu Gln Gly Tyr Leu Gln 785 790 795 800Lys Lys Val Arg Asp Gln Gln Leu Val Tyr Ser Ala Gly Val Tyr Arg 805 810815 Leu Pro Lys Asn Cys Ser 820 13 8 PRT Artificial sequence synthetic13 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5

We claim:
 1. A method of assaying abiquitin ligase activity comprising: a) combining: i) tag1-ubiquitin; ii) ubiquitin activating enzyme (E1); iii) ubiquitin conjugating enzyme (E2); iv) tag2-ubiquitin ligase (E3); b) measuring the amount of said tag1-ubiquitin bound to said ubiquitin ligase (E3), whereby the amount of said tag1-ubiquitin bound to said ubiquitin ligase (E3) indicates said ubiquitin ligase activity.
 2. The method of claim 1, further comprising: a) combining: v) a candidate ubiquitin ligase modulator.
 3. The method of claim 1, wherein said tag1 is a label or a partner of a binding pair.
 4. The method of claim 3, wherein said label is a fluorescent label.
 5. The method of claim 4, wherein said measuring is by measuring luminescence.
 6. The method of claim 3, wherein said partner of a binding pair is selected from the group consisting of an antigen, biotin, and calmodalin binding protein (CBP).
 7. The method of claim 6, wherein said partner of a binding pair is labeled by indirect labeling.
 8. The method of claim 7, wherein said indirect labeling is with a fluorescent label or a label enzyme.
 9. The method of claim 8, wherein said measuring is by measuring luminescence.
 10. The method of claim 8, wherein said label enzyme is selected from the group consisting of horseradish peroxidase, alkaline phosphatase and glucose oxidase.
 11. The method of claim 10, wherein said label enzyme is reacted with a label enzyme substrate which produces a fluorescent product.
 12. The method of claim 11, wherein said measuring is by measuring luminescence.
 13. The method of claim 12, wherein said tag1 is FALG (DYKDDDDK; SEG ID NO:13).
 14. The method of claim 8, wherein said partner of a binding pair is FLAG and said indirect labeling is via anti-FLAG (DYKDDDDK; SEG ID NO:13).
 15. The method of claim 7, wherein said partner of a binding pair is FLAG (DYKDDDDK; SEG ID NO:13).
 16. The method of claim 6, wherein said antigen is FLAG (DYKDDDDK; SEQ ID NO:13).
 17. The method of claim 3, wherein said tag2 is a surface substrate binding molecule.
 18. The method of claim 17, wherein said surface substrate binding molecule is a polyhistidine structure (His-tag).
 19. The method of claim 18, wherein said assaying is performed in a multi-well plate comprising a surface substrate comprising nickel. 